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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 towards sexually receptive (RF) over non-receptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERβ) expressed in the medial amygdala (MeA) in male preference towards RF. To further delineate the role played by ERβ in the MeA in the neuronal network regulating male preference, we developed a new ERβ-iCre mouse line using the CRISPR-Cas9 system. Fiber-photometry Ca2+imaging revealed that ERβ expressing neurons in the postero-dorsal part of the MeA (MeApd-ERβ+neurons) were more active during social investigation towards RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERβ+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-ERβ+neurons. Fiber-photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERβ+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-ERβ+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.Significance StatementIn this study, by introducing a new Cre mice line for ERβ+cells, we described the function of MeApd-ERβ+neurons and characteristics of their neuronal activity during preference tests. Using fiber photometry and DREADD techniques we have found MeApd-ERβ+neurons have a specific role in receptivity-based (receptive female vs. non-receptive female) preference but not in sexbased (receptive female vs. intact male) preference in male mice. We have also described this specific role of MeApd-ERβ+neurons is achieved by regulating the neuronal activity of downstream BNSTp neurons during receptivity-based, but not sex-based, preference tests. Our findings contribute to a better understating of the function of estrogen receptor expressing neurons in the neuronal network for the male-typical reproductive behaviors.

Oxytocin receptor (OXTR) modulates social behaviors in a species-specific manner. Remarkable inter- and intraspecies variation in brain OXTR distribution are associated with diversity in social behavior. To investigate potential genetic mechanisms underlying the phylogenetic plasticity in brainOxtrexpression and its consequences on social behavior, we constructed BAC transgenic mice harboring the entire prairie voleOxtrlocus with surrounding intergenic regulatory elements. Eight independent “volized” (pvOxtr) mouse lines were obtained; remarkably, each line displayed a unique pattern of brain expression distinct from mice and prairie voles. FourpvOxtrlines were selected for further investigation. Despite robust differences in brain expression,Oxtrexpression in mammary gland was conserved across lines, suggesting thatOxtrexpression in brain, but not mammary gland, is highly sensitive to local chromosomal landscape at integration sites. Moreover, different “volized” mouse lines showed differences in partner preference and maternal behaviors. Our results from this cross-species study suggest that species-specific variation in regulatory elements or distribution of transcription factors are not responsible for species-typical brainOxtrexpression patterns. Thus, transcriptional hypersensitivity to surrounding sequence of brainOxtrmay be a key mechanism to generate diversity in brain OXTR distribution and social behaviors. This inherent “evolvability” of brainOxtrexpression constitutes a novel transcriptional mechanism to generate variability in neuropeptide receptor distribution which, through natural selection, can generate diversity in adaptive social behaviors while preserving peripheral expression. The “volized”Oxtrmouse lines are useful for further understanding OXTR regulation and key neural circuits/networks mediating variability in social behaviors.Significance StatementUnlike the essential physiological phenomenon including feeding, sex, parturition and lactation, which are conserved across mammalian species, there is extraordinary diversity of social behaviors between and within species. Comparative studies across species suggest that variation in brain oxytocin receptor expression may mediate diversity in social behavior. Subtle variation in human oxytocin receptor sequence have been related to psychiatric phenotypes. Our studies suggest that the oxytocin receptor gene is hypersensitive to sequence variation at its molecular address which leads to variability in brain expression pattern and social behavior. This research provides new insights into the evolvability of genes producing diversity in social behaviors, which allows efficient adaptation of animals to variable environment and potentially provides insight into psychiatric outcomes related to social behavior.

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., Human Molecular Genetics, 30 (9), ISSN:0964-6906, ISSN:1460-2083

Estrogen receptors (ERα and ERβ) 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 ERβ change throughout life is unclear. Using genetically modified ERβ-RFP

Estrogens receptors (ER) are involved in several sociosexual behaviors and fear responses. In particular, the ERα is important for sexual behaviors, whereas ERβ modulates anxiolytic responses. Using shRNA directed either against the ERα or the ERβ 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 ERβ in this structure had no effect. This was also the case after silencing the ERα in the CeA. Silencing of the ERβ in this structure increased risk assessment, an expression of anxiety, and increased olfactory exploration of the environment. We hypothesize that the ERβ 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.

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 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.

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 patient’s families, clinicians, and the patients themselves. At the same time, 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 1β (IL-1β) in mediating individual differences in aggression using a resident-intruder mouse model. We found that non-aggressive mice exhibit higher levels of IL-1β 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 non-aggressive mice. In line with these findings, deletion of IL-1 receptor in the DRN caused enhanced c-Fos expression in 5-HT neurons during aggressive encounters, suggesting that modulation of 5-HT neuronal activity by IL-1β signaling in the DRN controls expression of aggressive behavior.

Post-translational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, more than 150 human GPI anchored proteins have been discovered and about 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 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 (KA)-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.

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), ERβ (βERKO), 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 β 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 βERKO mice remained similar to βWT. 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.

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

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.

It has long been known that the estrogen, 17β-estradiol (17β-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, 17β-E also regulates male-type sexual and aggressive behavior. In males, testosterone secreted from the testes is irreversibly aromatized to 17β-E in the brain. We discuss the contribution of two nuclear receptor isoforms, estrogen receptor (ER)α and ERβ to the estrogenic regulation of sexually dimorphic brain formation and sex-typical expression of these social behaviors. Furthermore, 17β-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 17β-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.

The present study investigated the role of oestrogen receptor (ER)α in the ventromedial nucleus of the hypothalamus (VMN), the preoptic area (POA), the medial amygdala (MePD) and the bed nucleus of stria terminalis (BNST) in sociosexual behaviour in female rats. This was conducted in two sets of experiments, with the VMN and POA investigated in the first set, and the MePD and BNST in the second set. The VMN and POA received intense projections from the MePD and BNST. We used a short hairpin RNA encoded within an adeno‐associated viral vector directed against the gene for ERα to reduce the number of ERα in the VMN or POA (first set of experiments) or in the BNST or MePD (second set of experiments) in female rats. The rats were housed in groups of four ovariectomised females and three males in a seminatural environment for 8 days. Compared with traditional test set‐ups, the seminatural environment provides an arena in which the rats can express their full behavioural repertoire, which allowed us to investigate multiple aspects of social and sexual behaviour in groups of rats. Behavioural observation was performed after oestrogen and progesterone injections. A reduction of ERα expression in the VMN or POA diminished the display of paracopulatory behaviours and lordosis responses compared to controls, whereas the lordosis quotient remained unaffected. This suggests that ERα in the VMN and POA play an important role in intrinsic sexual motivation. The reduction in ERα did not affect the social behaviour of the females, although the males sniffed and pursued the females with reduced ERα less than the controls. This suggests that the ERα in the VMN and POA is involved in the regulation of sexual attractiveness of females. The ERα in the MePD and BNST, on the other hand, plays no role in sociosexual behaviour.

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.

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.

The opioid peptides, dynorphin (DYN) and enkephalin (L-ENK) are contained in the hippocampal mossy fiber pathway where they modulate synaptic plasticity. In rats, the levels of DYN and L-ENK immunoreactivity (-ir) are increased when estrogen levels are elevated (Torres-Reveron et al. 2008 and 2009). Here, we used quantitative immunocytochemistry to examine whether opioid levels are similarly regulated in wildtype (WT) mice over the estrous cycle, and how these compared to males. Moreover, using estrogen receptor (ER) alpha and beta knockout mice (AERKO and BERKO, respectively), the present study examined the role of ERs in rapid, membrane-initiated (6 hr), or slower, nucleus-initiated (48 hr) estradiol effects on mossy fiber opioid levels. Unlike rats, the levels of DYN and L-ENK-ir did not change over the estrous cycle. However, compared to males, females had higher levels of DYN-ir in CA3a and L-ENK-ir in CA3b. In WT and BERKO ovariectomized (OVX) mice, neither DYN- nor L-ENK-ir changed following 6 or 48 hrs estradiol benzoate (EB) administration. However, DYN-ir significantly increased 48 hours after EB in the dentate gyrus (DG) and CA3b of AERKO mice only. These findings suggest that cyclic hormone levels regulate neither DYN nor L-ENK levels in the mouse mossy fiber pathway as they do in the rat. This may be due to species-specific differences in the mossy fiber pathway. However, in the mouse, DYN levels are regulated by exogenous EB in the absence of ERα possibly via an ERβ-mediated pathway requiring new gene transcription.

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.

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.

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.

Reproduction consists of various physiological events including fertilization, development of sexual characteristics, social behavior, maturation, and aging. Reproductive functions are ultimately regulated by the hypothalamus-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neuropeptide that regulates vertebrate reproduction (Schally et al., 1972). In tetrapods, GnRH neurons are located in the preoptic-hypothalamic region and project their fibers to the median eminence to regulate gonadotropin secretion from the anterior pituitary gland, which stimulates sex steroid secretion and gametogenesis in the gonads. It was also shown that central administration of GnRH can stimulate female sexual behavior in rats (Moss and McCann, 1973; Pfaff, 1973). GnRH release is regulated by other neuropeptides, neurotransmitters, and steroid hormones. Watanabe et al. summarize the role of gamma-amino butyric acid (GABA) in the regulation of GnRH neuronal activity and discuss functional consequences of GABAergic inputs to GnRH neurons in physiological aspects of reproduction. Recently, two neuropeptides containing the C-terminal Arg-Phe-NH2 (RFamide peptides), kisspeptin, and gonadotropin-inhibitory hormone (GnIH), emerged as critical accelerator, and suppressor, respectively, of vertebrate reproduction. Parhar et al. highlight classical and recent findings regarding the role of GnRH, kisspeptin, and GnIH in the regulation of social behaviors in fish, birds, and mammals, and discuss their importance in future biological and biomedical researches (Perspectives).

Oestradiol exerts a profound influence upon multiple brain circuits. For the most part, these effects are mediated by oestrogen receptor (ER)α. We review here the roles of ERβ, the other ER isoform, in mediating rodent oestradiol-regulated anxiety, aggressive and sexual behaviours, the control of gonadotrophin secretion, and adult neurogenesis. Evidence exists for: (i) ERβ located in the paraventricular nucleus underpinning the suppressive influence of oestradiol on the stress axis and anxiety-like behaviour; (ii) ERβ expressed in gonadotrophin-releasing hormone neurones contributing to oestrogen negative-feedback control of gonadotrophin secretion; (iii) ERβ controlling the offset of lordosis behaviour; (iv) ERβ suppressing aggressive behaviour in males; (v) ERβ modulating responses to social stimuli; and (vi) ERβ in controlling adult neurogenesis. This review highlights two major themes; first, ERβ and ERα are usually tightly inter-related in the oestradiol-dependent control of a particular brain function. For example, even though oestradiol feedback to control reproduction occurs principally through ERα-dependent mechanisms, modulatory roles for ERβ also exist. Second, the roles of ERα and ERβ within a particular neural network may be synergistic or antagonistic. Examples of the latter include the role of ERα to enhance, and ERβ to suppress, anxiety-like and aggressive behaviours. Splice variants such as ERβ2, acting as dominant negative receptors, are of further particular interest because their expression levels may reflect preceeding oestradiol exposure of relevance to oestradiol replacement therapy. Together, this review highlights the predominant modulatory, but nonetheless important, roles of ERβ in mediating the many effects of oestradiol upon adult brain function.

To investigate the effects of early life stress on the development of social behaviors in male mice, we examined behavioral responses toward same sex stimulus mice in the social investigation test and aggressive behaviors in peripubertal male mice exposed to maternal separation (MS) during the first 2 weeks of life. MS suppressed aggressive behaviors from 5-9 weeks of age, but had no effect on social investigative behaviors in the social investigation test. Investigation of neuroendocrine bases of behavioral effects of MS showed that MS reduced plasma testosterone levels and decreased arginine vasopressin and increased oxytocin immunoreactivity in the paraventricular nucleus of peripubertal males. These results collectively suggest that early life stress disrupts the development of male aggressive behaviors and associated neuroendocrine systems.

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.

This article describes background information for the symposium held at the 70th Annual meeting of the Japanese Society for Animal Psychology. The main theme of this symposium was to provide updated information on neurodevelopmental toxicity caused by environmental chemicals, through lectures by three distinguished researchers, and to discuss possible contributions and expected roles of animal psychology in toxicological studies designed to protect children's health. The Ministry of the Environment has commenced a large-scale, long-term epidemiological study entitled the “Environment and Children's Study (Eco-Chil)”, to elucidate health effects caused by chemical exposures of children including pervasive developmental disorders. Neurobehavioral assessments have been included in globally accepted guidelines for neurotoxicity and reproductive toxicity studies in rodents as effective tools for collecting experimental evidence. Findings from these experiments provide a scientific basis for the epidemiological studies, like Eco-Chil. However, currently available behavioral testing methods are insufficient for detecting neurodevelopmental effects of environmental chemicals, especially for emotionality and social aspects of behavior. Appropriate animal behavioral models are needed to advance toxicological studies further. Animal psychologists could greatly contribute to the development of these animal models and the interpretation of behavioral test results.

Stress is one of the important factors to activate the sympathetic nervous system. We recently reported that central administration of corticotropin-releasing factor (CRF), known as a stress-related neuropeptide, increases the expression of both cyclooxygenase (COX) and nitric oxide synthase (NOS) in presympathetic neurons in the paraventricular hypothalamic nucleus (PVN). In the present study, therefore, we investigated whether brain COX and NOS can also mediate restraint stress (RS)-induced sympathetic activation by assessing the plasma catecholamine levels and neuronal activation of presympathetic neurons in the PVN. In addition, we examined effects of RS on the expression of both COX and NOS isozymes in the presympathetic PVN neurons. Intraperitoneal administration of an inhibitor for COX-1, COX-2 or inducible NOS (iNOS), but not for neuronal NOS (nNOS), reduced RS-induced elevation of plasma catecholamine levels and Fos expression in the presympathetic PVN neurons. Moreover, RS increased the expression of COX-1, COX-2 and iNOS in the presympathetic PVN neurons, whereas nNOS expression did not change. These results suggest that COX-1, COX-2 and iNOS in the presympathetic PVN neurons mediate acute RS-induced sympathetic activation.

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.

Eating is modulated by estradiol in females of many species and in women. To further investigate the estrogen receptor mechanism mediating this effect, ovariectomized rats and mice were treated with estradiol benzoate or the estrogen receptor-alpha (ER-alpha)-selective agonist PPT. PPT inhibited eating in rats much more rapidly than estradiol (approximately 2-6 h versus >24 h). In contrast, the latencies to vaginal estrus after PPT and estradiol were similar (>24 h). PPT also inhibited eating within a few hours in wild-type mice, but failed to inhibit eating in transgenic mice deficient in ER-alpha (ERalphaKO mice). PPT, but not estradiol, induced the expression of c-Fos in corticotrophin-releasing hormone (CRH)-expressing cells of the paraventricular nucleus (PVN) of the hypothalamus within 90-180 min in rats. Both PPT and estradiol reduced c-Fos expression in an ER-alpha-containing area of the nucleus of the solitary tract. The anomalously rapid eating-inhibitory effect of PPT suggests that PPT's neuropharmacological effect differs from estradiol's, perhaps because PPT differentially activates membrane versus nuclear ER-alpha or because PPT activates non-ER-alpha membrane estrogen receptors in addition to ER-alpha. The failure of PPT to inhibit eating in ERalphaKO mice, however, indicates that ER-alpha is necessary for PPT's eating-inhibitory action and that any PPT-induced activation of non-ER-alpha estrogen receptors is not sufficient to inhibit eating. Finally, the rapid induction of c-Fos in CRH-expressing cells in the PVN by PPT suggests that PPT elicits a neural response that is similar to that elicited by stress or aversive emotional stimuli.

It has been shown that long-term estrogen treatment in gonadectomized female mice increases anxiety levels. On the other hand, a recent study has reported that estrogen may down-regulate the levels of anxiety by acting through estrogen receptor (ER) beta. In the present study, we investigated the role of ER-beta in the regulation of anxiety levels in female mice after long-term estrogen treatment. Gonadectomized ER-beta knockout (betaERKO) female mice and their wild type (betaWT) littermates were implanted several different doses (experiment 1: 2.0 microg/day, experiment 2: 1.0, 0.4, 0.2 or 0.1 microg/day) of an estradiol benzoate (EB) or placebo pellet. Ten days after pellet implant, behavioral tests commenced to measure the anxiety levels (experiment 1: light-dark transition test (LDT), experiment 2: LDT, elevated plus maze test (EPM) and social investigation test (SIT)). We found that, at higher-doses, long-term treatment of EB had anxiogenic effects in both betaWT and betaERKO mice as indicated by a decrease of the time spent in the light side and the number of transitions between two sides during LDT. In contrast, several behavioral measurements indicated that the lower-doses treatment of EB might reduce the anxiety levels possibly through ER-beta. Particularly, the anxiolytic effects of EB in the SIT were more pronounced in betaWT mice than betaERKO mice. Together, the findings in the present study suggest that estrogen may have both anxiolytic and anxiogenic effects in female mice, and that ER-beta gene disruption did not affect anxiogenic regulation by estrogen in female mice, but partially affected anxiolytic regulation.

application/pdf, The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17β-estradiol (E2) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E2, with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER){alpha} and ERβ were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ER{alpha} mediates E2 action on PRL cells. A few PRL cells were observed in ER{alpha}-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E2 on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E2. The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.

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.

Estrogen receptor alpha (ERalpha) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERalpha signaling in ERalpha knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERalpha in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERalpha expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERalpha levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERalpha in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.

Genes are not expressed in isolation any more than social behavior has meaning outside of society. Both are in dynamic flux with the immediate environment that the gene/individual finds itself, which in turn establishes the timing, pattern, and conditions of expression. This means that complex behaviors and their genetic underpinnings should be viewed as a cumulative process, or as the result of experiences up to that point in time and, at the same time, as setting the stage for what will follow. The evidence indicates that as experiences accumulate throughout life, early experiences shape how genes/individuals will respond to later experiences, whereas later experiences modify the effects of these earlier experiences. A method of graphically representing and analyzing change in gene and neural networks is presented. Results from several animal model systems will be described to illustrate these methods. First, we will consider the phenomenon of temperature-dependent sex determination in reptiles. We will illustrate how the experience of a particular temperature during a sensitive period of embryogenesis sculpts not only the patterns of expression of genes involved in sex determination and gonadal differentiation but also the morphological, physiological, neuroendocrine, and behavioral traits of the adult phenotype. The second model system concerns the effects of the sex ratio in the litter in rats, and the genotype ratio in the litter of transgenic mice, on the nature and frequency of maternal care and how this in turn influences the patterns of activation of identified neural circuits subserving the offspring's sociosexual behavior when it is an adult.

A major cost of social behaviour is the increased risk of exposure to parasites. Female mice discriminate between uninfected males and males infected with parasites such as the intestinal nematode Heligmosomoides polygyrus, on the basis of social cues, displaying aversive responses to the odours of infected males and preferentially mating with nonparasitized males. Here, using female mice whose gene for the neuropeptide, oxytocin (OT), has been deleted (OT knockout mice, OTKO), we show that at least one normal allele for OT is required for the discrimination, individual recognition and avoidance of H. polygyrus parasitized males on the basis of odour. In an odour choice test, female wild-type (OTWT) and heterozygous (OTHT) mice displayed a marked initial choice for the odours of uninfected males over infected males that were either familiar or novel, whereas the OTKO females showed no consistent initial choice. This was not associated with olfactory deficits because females of all three genotypes distinguished between the odours of uninfected males of varying sexual states. A 1-min exposure to the odours of infected males also induced analgesic responses (i.e. pain inhibition) in OTWT and OTHT females, with the OTKO females displaying a markedly attenuated analgesia. These analgesic responses and the associated behavioural correlates of anxiety/fear facilitate the expression of a reduced interest in and avoidance of parasitized males by females. The OTWT and OTHT, but not the OTKO females, also distinguished between the odours of novel and familiar infected males and modulated their analgesic responses on the basis of prior familiarity, displaying reduced analgesia to familiar males and enhanced analgesia to novel males. Our findings indicate that a normal OT gene is an essential part of the central mechanism whereby females can, on the basis of odour, discriminate between parasitized and nonparasitized males and, thereby, both reduce the transmission of parasites to themselves and select for parasite-free males.