Central oxytocin receptors mediate mating-induced partner preferences and enhance correlated activation across forebrain nuclei in male prairie voles

Oxytocin (OT) is a deeply conserved nonapeptide that acts both peripherally and centrally to modulate reproductive physiology and sociosexual behavior across divergent taxa, including humans. In vertebrates, the distribution of the oxytocin receptor (OTR) in the brain is variable within and across species, and OTR signaling is critical for a variety of species-typical social and reproductive behaviors, including affiliative and pair bonding behaviors in multiple socially monogamous lineages of fishes, birds, and mammals. Early work in prairie voles suggested that the endogenous OT system modulates mating-induced partner preference formation in females but not males; however, there is significant evidence that central OTRs may modulate pair bonding behavior in both sexes. In addition, it remains unclear how transient windows of central OTR signaling during sociosexual interaction modulate neural activity to produce enduring shifts in sociobehavioral phenotypes, including the formation of selective social bonds. Here we re-examine the role of the central OT system in partner preference formation in male prairie voles using a selective OTR antagonist delivered intracranially. We then use the same antagonist to examine how central OTRs modulate behavior and immediate early gene (Fos) expression, a metric of neuronal activation, in males during brief sociosexual interaction with a female. Our results suggest that, as in females, OTR signaling is critical for partner preference formation in males and enhances correlated activation across sensory and reward processing brain areas during sociosexual interaction. These results are consistent with the hypothesis that central OTR signaling facilitates social bond formation by coordinating activity across a pair bonding neural network.     

Sex-specific activity and function of hypothalamic nonapeptide neurons during nest-building in zebra finches

Vertebrate species from fish to humans engage in a complex set of preparatory behaviors referred to as nesting; yet despite its phylogenetic ubiquity, the physiological and neural mechanisms that underlie nesting are not well known. We here test the hypothesis that nesting behavior is influenced by the vasopressin–oxytocin (VP–OT) peptides, based upon the roles they play in parental behavior in mammals. We quantified nesting behavior in male and female zebra finches following both peripheral and central administrations of OT and V_1a receptor (OTR and V1aR, respectively) antagonists. Peripheral injections of the OTR antagonist profoundly reduce nesting behavior in females, but not males, whereas comparable injections of V1aR antagonist produce relatively modest effects in both sexes. However, central antagonist infusions produce no effects on nesting, and OTR antagonist injections into the breast produce significantly weaker effects than those into the inguinal area, suggesting that antagonist effects are mediated peripherally, likely via the oviduct. Finally, immunocytochemistry was used to quantify nesting-induced Fos activation of nonapeptide neurons in the paraventricular and supraoptic nuclei of the hypothalamus and the medial bed nucleus of the stria terminalis. Nest-building induced Fos expression within paraventricular VP neurons of females but not males. Because the avian forms of OT (Ile⁸-OT; mesotocin) and VP (Ile³-VP; vasotocin) exhibit high affinity for the avian OTR, and because both peptide forms modulate uterine contractility, we hypothesize that nesting-related stimuli induce peptide release from paraventricular vasotocin neurons, which then promote female nesting via peripheral feedback from OTR binding in the oviduct uterus.     

Manipulation of the oxytocin system alters social behavior and attraction in pair-bonding primates, Callithrix penicillata

The establishment and maintenance of stable, long-term male-female relationships, or pair-bonds, are marked by high levels of mutual attraction, selective preference for the partner, and high rates of sociosexual behavior. Central oxytocin (OT) affects social preference and partner-directed social behavior in rodents, but the role of this neuropeptide has yet to be studied in heterosexual primate relationships. The present study evaluated whether the OT system plays a role in the dynamics of social behavior and partner preference during the first 3 weeks of cohabitation in male and female marmosets, Callithrix penicillata. OT activity was stimulated by intranasal administration of OT, and inhibited by oral administration of a non-peptide OT-receptor antagonist (L-368,899; Merck). Social behavior throughout the pairing varied as a function of OT treatment. Compared to controls, marmosets initiated huddling with their social partner more often after OT treatments but reduced proximity and huddling after OT antagonist treatments. OT antagonist treatment also eliminated food sharing between partners. During the 24-h preference test, all marmosets interacted more with an opposite-sex stranger than with the partner. By the third-week preference test, marmosets interacted with the partner and stranger equally with the exception that intranasal-OT treatments facilitated initial partner-seeking behavior over initial contact with the stranger. Our findings demonstrate that pharmacological manipulations of OT activity alter partner-directed social behavior during pair interactions, suggesting that central OT may facilitate the process of pair-bond formation and social relationships in marmoset monkeys.     

Beta 2-agonist treatment enhances uterine oxytocin receptor mRNA expression in pregnant rats

The objective of this study was to disclose an interaction between Beta(2)-adrenergic (Beta(2)-ARs) and oxytocin (OT) receptors (OTRs) in the late-pregnant rat uterus. We investigated the level of uterine OTR mRNA expression after the administration of Beta(2)-AR agonists fenoterol and hexoprenaline to rats from day 18 to 22 of pregnancy, and also tested the effect of fenoterol on uterine explants. Hexoprenaline induced a maximum 24% increase of OTR mRNA. Fenoterol in vivo elicited a maximum 125% increase of OTR mRNA, in vitro produced a maximum fourfold increase in OTR mRNA. In fenoterol-treated rats the maximal contractility increasing effect of OT on isolated uterine rings was significantly higher than in intact term pregnant rats, but the EC50 values were not statistically different. It was concluded that the enhanced expression of OTR mRNA induced by Beta(2)-agonists in the late-pregnant rat uterus may be a possible drawback to effective therapy of preterm uterine contractions with Beta(2)-agonists.     

Agonist-specific, high-affinity binding epitopes are contributed by an arginine in the N-terminus of the human oxytocin receptor

The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding. The aim of this study was to identify which individual residues within the N-terminal domain of the human OTR provided these OT binding epitopes. A series of truncated OTRs and mutant receptor constructs with systematic alanine substitution were characterized with respect to their pharmacological profile and intracellular signaling capability. Although a number of residues within the OTR will be required for optimal OT-OTR interaction, our data establish that Arg(34) within the N-terminal domain contributes to high-affinity OT binding. Removal of Arg(34) by truncation or substitution resulted in a 2000-fold decrease in OT affinity. In addition, we show that the arginyl at this locus is required for high-affinity binding of agonists in general. However, the importance of Arg(34) is restricted to agonist interaction with the OTR, as it was not required for binding peptide antagonist or non-peptide antagonist. It is noteworthy that the corresponding Arg in the related rat V(1a) vasopressin receptor is also required for high-affinity agonist binding. This study defines, at the molecular level, the role of the N-terminus of the OTR in high-affinity agonist binding and identifies a key residue for this function.     

Central blockade of oxytocin receptors during mid-late gestation reduces amplitude of slow afterhyperpolarization in supraoptic oxytocin neurons

The neurohypophysial hormone oxytocin (OT), synthesized in magnocellular paraventricular (PVN) and supraoptic (SON) nuclei, is well known for its effects in lactation. Our previous studies showed that central OT receptor (OTR) binding is increased during gestation and that blockade of central OTRs, specifically during mid-late gestation, causes a delay in OT release during suckling and reduces weight gain in pups, suggesting decreased milk delivery. In the present study, we tested whether central OTR blockade during late gestation disrupts the gestation-related plasticity in intrinsic membrane properties. Whole cell current-clamp recordings were performed in OT neurons from pregnant rats (19-22 days in gestation) that were infused with an OTR antagonist (OTA) or artificial cerebrospinal fluid (aCSF) and from virgin rats infused with aCSF into the third ventricle via an osmotic minipump beginning on days 12-14 of gestation. The amplitudes of both Ca(2+)-dependent afterhyperpolarizations (AHPs), an apamin-sensitive medium AHP (mAHP) and an apamin-insensitive slow AHP (sAHP), were significantly increased during late gestation in control pregnant animals. However, the amplitude of the sAHP from pregnant rats treated with the OTA was significantly smaller than that of pregnant control rats and similar to that of virgins. These results indicate that the diminished efficiency in lactation due to OTR blockade may be partly a result of an altered sAHP that would shape OT bursting. These findings suggest that central actions of OT during late gestation are necessary for programming the plasticity of at least some of the intrinsic membrane properties in OT neurons during lactation.     

Sex differences in oxytocin receptor binding in forebrain regions: Correlations with social interest in brain region- and sex- specific ways

Social interest reflects the motivation to approach a conspecific for the assessment of social cues and is measured in rats by the amount of time spent investigating conspecifics. Virgin female rats show lower social interest towards unfamiliar juvenile conspecifics than virgin male rats. We hypothesized that the neuropeptide oxytocin (OT) may modulate sex differences in social interest because of the involvement of OT in pro-social behaviors. We determined whether there are sex differences in OT system parameters in the brain and whether these parameters would correlate with social interest. We also determined whether estrus phase or maternal experience would alter low social interest and whether this would correlate with changes in OT system parameters. Our results show that regardless of estrus phase, females have significantly lower OT receptor (OTR) binding densities than males in the majority of forebrain regions analyzed, including the nucleus accumbens, caudate putamen, lateral septum, bed nucleus of the stria terminalis, medial amygdala, and ventromedial hypothalamus. Interestingly, male social interest correlated positively with OTR binding densities in the medial amygdala, while female social interest correlated negatively with OTR binding densities in the central amygdala. Proestrus/estrus females showed similar social interest to non-estrus females despite increased OTR binding densities in several forebrain areas. Maternal experience had no immediate or long-lasting effects on social interest or OT brain parameters except for higher OTR binding in the medial amygdala in primiparous females. Together, these findings demonstrate that there are robust sex differences in OTR binding densities in multiple forebrain regions of rats and that OTR binding densities correlate with social interest in brain region- and sex-specific ways.     

Developmental effects of oxytocin on neural activation and neuropeptide release in response to social stimuli

Previous studies have revealed that the neuropeptide hormone oxytocin (OT) has developmental effects on subsequent social behavior and on mechanisms underlying social behavior such as OT neurons and estrogen receptor alpha. This suggests that OT might also have developmental effects on neural responses to social stimuli. This was tested in socially monogamous prairie voles (Microtus ochrogaster) by manipulating OT on the first day of life and then assessing the response to a heterosexual pairing in adulthood. The response to cohabitation was assessed by quantifying neural activation in regions of the brain associated with sociosexual behavior and anxiety using c-Fos immunoreactivity. Additionally, immunocytochemistry was used to label OT and vasopressin neurons and plasma was assayed for both neuropeptides. Treatment effects were evident in females, but not in males. Blockade of OT receptors with an OT antagonist on the first day of life resulted in neural activation of the central amygdala in response to a pairing with a novel male in adulthood. The central amygdala does not normally express c-Fos after a heterosexual pairing in reproductively na?ve prairie voles. Treatment effects also were observed in vasopressin immunoreactivity in the SON with OT-treated females showing a decrease.     

Oxytocin (OT) and arginine-vasopressin (AVP) act on OT receptors and not AVP V1a receptors to enhance social recognition in adult Syrian hamsters (Mesocricetus auratus)

Social recognition is a fundamental requirement for all forms of social relationships. A majority of studies investigating the neural mechanisms underlying social recognition in rodents have investigated relatively neutral social stimuli such as juveniles or ovariectomized females over short time intervals (e.g., 2 h). The present study developed a new testing model to study social recognition among adult males using a potent social stimulus. Flank gland odors are used extensively in social communication in Syrian hamsters and convey important information such as dominance status. We found that the recognition of flank gland odors after a 3 min exposure lasted for at least 24 h, substantially longer than the recognition of other social cues in rats and mice. Intracerebroventricular injections of OT and AVP prolonged the recognition of flank gland odor for up to 48 h. Selective OTR but not V1aR agonists, mimicked these enhancing effects of OT and AVP. Similarly, selective OTR but not V1aR antagonists blocked recognition of the odors after 20 min. In contrast, the recognition of non-social stimuli was not blocked by either the OTR or the V1aR antagonists. Our findings suggest both OT and AVP enhance social recognition via acting on OTRs and not V1aRs and that the recognition enhancing effects of OT and AVP are limited to social stimuli.     

Endogenous oxytocin is necessary for preferential Fos expression to male odors in the bed nucleus of the stria terminalis in female Syrian hamsters

Successful reproduction in mammals depends on proceptive or solicitational behaviors that enhance the probability of encountering potential mates. In female Syrian hamsters, one such behavior is vaginal scent marking. Recent evidence suggests that the neuropeptide oxytocin (OT) may be critical for regulating this behavior. Blockade of OT receptors in the bed nucleus of the stria terminalis (BNST) or the medial preoptic area (MPOA) decreases vaginal marking responses to male odors; lesion data suggest that BNST, rather than MPOA, mediates this effect. However, how OT interacts with sexual odor processing to drive preferential solicitation is not known. To address this issue, intact female Syrian hamsters were exposed to male or female odors and their brains processed for immunohistochemistry for Fos, a marker of recent neuronal activation, and OT. Additional females were injected intracerebroventricularly (ICV) with an oxytocin receptor antagonist (OTA) or vehicle, and then tested for vaginal marking and Fos responses to sexual odors. Colocalization of OT and Fos in the paraventricular nucleus of the hypothalamus was unchanged following exposure to male odors, but decreased following exposure to female odors. Following injections of OTA, Fos expression to male odors was decreased in BNST, but not in MPOA or the medial amygdala (MA). Fos expression in BNST may be functionally relevant for vaginal marking, given that there was a positive correlation between Fos expression and vaginal marking for BNST, but not MPOA or MA. Together, these data suggest that OT facilitation of neuronal activity in BNST underlies the facilitative effects of OT on solicitational responses to male odors.     

Oxytocin in the medial preoptic area facilitates male sexual behavior in the rat

Oxytocin (OT) is a versatile neuropeptide that is involved in a variety of mammalian behaviors, and its role in reproductive function and behavior has been well established. The majority of pharmacological studies of the effects of OT on male sexual behavior have focused on the paraventricular nucleus (PVN), ventral tegmental area (VTA), hippocampus, and amygdala. Less attention has been given to the medial preoptic area (MPOA), a major integrative site for male sexual behavior. The present study investigated the effects of intra-MPOA administration of OT and (d(CH2)51, Tyr(Me)2, Thr4, Orn8, Tyr-NH29)-vasotocin, an OT antagonist (OTA), on copulation in the male rat. The relationship between OT receptor (OTR) binding levels in the MPOA and sexual efficiency was also explored. Microinjection of OT into the MPOA facilitated copulation in sexually experienced male rats, whereas similar injections of an OTA inhibited certain aspects of copulation but had no significant effect on locomotor activity in an open field. Contrary to expectation, sexually efficient males had lower levels of OTR binding in the rostral MPOA compared to inefficient animals. The present data suggest that OT activity in the MPOA is not necessary for the expression of male sexual behavior but is sufficient to facilitate copulatory behaviors and improve sexual efficiency in sexually experienced male rats. These data also suggest that OTR activity in the MPOA stimulates anogenital investigation, facilitates the initiation of copulation, and plays a role in the sensitization effect of the first ejaculation on subsequent ejaculations.     

Species and individual differences in juvenile female alloparental care are associated with oxytocin receptor density in the striatum and the lateral septum

The neuropeptide oxytocin has been implicated in the regulation of affiliative behavior and maternal responsiveness in several mammalian species. Rodent species vary considerably in the expression of juvenile alloparental behavior. For example, alloparental behavior is spontaneous in juvenile female prairie voles (approximately 20 days of age), takes 1-3 days of pup exposure to develop in juvenile rats, and is nearly absent in juvenile mice and meadow voles. Here, we tested the hypothesis that species differences in pup responsiveness in juvenile rodents are associated with oxytocin receptor (OTR) density in specific brain regions. We found that OTR density in the nucleus accumbens (NA) is highest in juvenile prairie voles, intermediate in juvenile rats, and lowest in juvenile mice and meadow voles. In the caudate putamen (CP), OTR binding was highest in prairie voles, intermediate in rats and meadow voles, and lowest in mice. In contrast, the lateral septum (LS) shows an opposite pattern, with OTR binding being high in mice and meadow voles and low in prairie voles and rats. Thus, alloparental responsiveness in juvenile rodents is positively correlated with OTR density in the NA and CP and negatively correlated with OTR density in the LS. We then investigated whether a similar receptor-behavior relationship exists among juvenile female prairie voles by correlating individual variation in alloparental behavior with variation in OTR density. The time spent adopting crouching postures, the most distinctive component of alloparental behavior in juveniles, was positively correlated with OTR density in the NA (r = 0.47) and CP (r = 0.45) and negatively correlated with OTR density in the lateral septum (r = -0.53). Thus, variation in OTR density in the NA, CP, and LS may underlie both species and individual differences in alloparental care in rodents.     

Neonatal paternal deprivation impairs social recognition and alters levels of oxytocin and estrogen receptor α mRNA expression in the MeA and NAcc,and serum oxytocin in mandarin voles

Paternal care is necessary for the healthy development of social behavior in monogamous rodents and social recognition underpins social behavior in these animals. The effects of paternal care on the development of social recognition and underlying neuroendocrine mechanisms, especially the involvement of oxytocin and estrogen pathways, remain poorly understood. We investigated the effects of paternal deprivation (PD: father was removed from neonatal pups and mother alone raised the offspring) on social recognition in mandarin voles (Microtus mandarinus), a socially monogamous rodent. Paternal deprivation was found to inhibit the development of social recognition in female and male offspring according to a habituation–dishabituation paradigm. Paternal deprivation resulted in increased inactivity and reduced investigation during new encounters with other animals. Paternal deprivation reduced oxytocin receptor (OTR) and estrogen receptor α (ERα) mRNA expression in the medial amygdala and nucleus accumbens. Paternal deprivation reduced serum oxytocin (OT) concentration in females, but had no effect on males. Our results provide substantial evidence that paternal deprivation inhibits the development of social recognition in female and male mandarin voles and alters social behavior later in life. This is possibly the result of altered expression of central OTR and ERα and serum OT levels caused by paternal deprivation.     

Mountain gorilla life histories,reproductive competition,and sociosexual behavior and some implications for captive husbandry

New data on modal patterns of, and variability in, mountain gorilla life history tactics and sociosexual behavior, collected during long-term fieldwork at the Karisoke Research Centre, are presented here. These data show that immature males and females develop sociosexual relationships with both peers and adults, and that these relationships—which contribute to more complex social relationships—often lead to mating between natal females and males who are “familiar” partners. They also show that within-group reproductive competition between males can reach considerable levels, but that immature males are sometimes able to copulate with both nulliparous and parous females; that unrelated silverbacked males can reside in the same all-male group but become intolerant of each other if females join the group; and that, whereas females sometimes compete with each other for opportunities of copulate, their probability of conceiving is probably not decreased by any inability to control the mating situation. This information may contribute to continued improvement in the husbandry of captive gorillas.     

The cross-generation transmission of oxytocin in humans

Animal studies demonstrated that the neuropeptide oxytocin (OT), implicated in bond formation across mammalian species, is transmitted from mother to young through mechanisms of early social experiences; however, no research has addressed the cross-generation transmission of OT in humans. Fifty-five parents (36 mothers and 19 fathers) engaged in a 15-min interaction with their infants. Baseline plasma OT was sampled from parents and salivary OT was sampled from parents and infants before and after play and analyzed with ELISA methods. Interactions were micro-coded for parent and child's socio-affective behavior. Parent and infant's salivary OT was individually stable across assessments and showed an increase from pre- to post-interaction. Significant correlations emerged between parental and infant OT at both assessments and higher OT levels in parent and child were related to greater affect synchrony and infant social engagement. Parent-infant affect synchrony moderated the relations between parental and infant OT and the associations between OT in parent and child were stronger under conditions of high affect synchrony. Results demonstrate consistency in the neuroendocrine system supporting bond formation in humans and other mammals and underscore the role of early experience in shaping the cross-generation transmission of social affiliation in humans.     

The oxytocin receptor antagonist atosiban inhibits cell growth via a "biased agonist" mechanism

In human myometrial cells, the promiscuous coupling of the oxytocin receptors (OTRs) to G(q) and G(i) leads to contraction. However, the activation of OTRs coupled to different G protein pathways can also trigger opposite cellular responses, e.g. OTR coupling to G(i) inhibits, whereas its coupling to G(q) stimulates, cell proliferation. Drug analogues capable of promoting a selective receptor-G protein coupling may be of great pharmacological and clinical importance because they may target only one specific signal transduction pathway. Here, we report that atosiban, an oxytocin derivative that acts as a competitive antagonist on OTR/G(q) coupling, displays agonistic properties on OTR/G(i) coupling, as shown by specific (35)S-labeled guanosine 5'-3-O-(thio) trisphosphate ([(35)S]GTPgammaS) binding. Moreover, atosiban, by acting on a G(i)-mediated pathway(,) inhibits cell growth of HEK293 and Madin-Darby canine kidney cells stably transfected with OTRs and of DU145 prostate cancer cells expressing endogenous OTRs. Notably, atosiban leads to persistent ERK1/2 activation and p21(WAF1/CIP1) induction, the same signaling events leading to oxytocin-mediated cell growth inhibition via a G(i) pathway. Finally, atosiban exposure did not cause OTR internalization and led to only a modest decrease (20%) in the number of high affinity cell membrane OTRs, two observations consistent with the finding that atosiban did not lead to any desensitization of the oxytocin-induced activation of the G(q)-phospholipase C pathway. Taken together, these observations indicate that atosiban acts as a "biased agonist" of the human OTRs and thus belongs to the class of compounds capable of selectively discriminating only one among the multiple possible active conformations of a single G protein-coupled receptor, thereby leading to the selective activation of a unique intracellular signal cascade.     

Fibroblast growth factor 2 alters the oxytocin receptor in a developmental model of anxiety-like behavior in male rat pups

We aimed to determine the short-term effects of early-life stress in the form of maternal separation (MS) on anxiety-like behavior in male rat pups. In order to assess anxiety, we measured 40 kHz separation-induced ultrasonic vocalizations (USV) on postnatal day (PND) 11. We further aimed to evaluate the potential involvement of two neurochemical systems known to regulate social and anxiety-like behaviors throughout life: oxytocin (OT) and fibroblast growth factor 2 (FGF2). For these purposes, we tested the effects of neonatal administration (on PND1) of an acute dose of FGF2 on USV and its potential interaction with MS. In addition, we validated the anxiolytic effects of OT and measured oxytocin receptor (OTR) gene expression, binding and epigenetic regulation via histone acetylation. Our results show that MS potentiated USV while acute administration of OT and FGF2 attenuated them. Further, we found that both FGF2 and MS increased OTR gene expression and the association of acH3K14 with the OTR promoter in the bed nucleus of the stria terminalis (BNST). Comparable changes, though not as pronounced, were also found for the central amygdala (CeA). Our findings suggest that FGF2 may exert its anxiolytic effects in male MS rats by a compensatory increase in the acetylation of the OTR promoter to overcome reduced OT levels in the BNST.     

Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERalpha) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of the following treatments: (a) 50microl i.p. injection of 7microg OT; (b) 0.7microg of OT antagonist (OTA); or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERalpha and estrogen receptor beta (ERbeta) were compared by age, treatment, and sex utilizing real time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERalpha increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21, there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERalpha mRNAs and that these effects are mitigated by D21. Also with the exception of ERalpha mRNA, the effects are the same in both sexes.