Monitoring oxytocin signaling in the brain: More than a love story.

More than any other neuropeptide, oxytocin (OXT) is attracting the attention of neurobiologists, psychologists, psychiatrists, evolutionary biologists and even economists. It is often called a "love hormone" due to its many prosocial functions described in vertebrates including mammals and humans, especially its ability to support "bonding behaviour". Oxytocin plays an important role in female reproduction, as it promotes labour during parturition, enables milk ejection in lactation and is essential for related reproductive behaviours. Therefore, it particularly attracts the interest of many female researchers. In this short narrative review I was invited to provide a personal overview on my scientific journey closely linked to my research on the brain OXT system and the adventures associated with starting my research career behind the Iron Curtain.

Functional involvement of septal miR-132 in extinction and oxytocin-mediated reversal of social fear.

Social interactions are critical for mammalian survival and evolution. Dysregulation of social behavior often leads to psychopathologies such as social anxiety disorder, denoted by intense fear and avoidance of social situations. Using the social fear conditioning (SFC) paradigm, we analyzed expression levels of miR-132-3p and miR-124-3p within the septum, a brain region essential for social preference and avoidance behavior, after acquisition and extinction of social fear. Here, we found that SFC dynamically altered both microRNAs. Functional in vivo approaches using pharmacological strategies, inhibition of miR-132-3p, viral overexpression of miR-132-3p, and shRNA-mediated knockdown of miR-132-3p specifically within oxytocin receptor-positive neurons confirmed septal miR-132-3p to be critically involved not only in social fear extinction, but also in oxytocin-induced reversal of social fear. Moreover, Argonaute-RNA-co-immunoprecipitation-microarray analysis and further in vitro and in vivo quantification of target mRNA and protein, revealed growth differentiation factor-5 (Gdf-5) as a target of miR-132-3p. Septal application of GDF-5 impaired social fear extinction suggesting its functional involvement in the reversal of social fear. In summary, we show that septal miR-132-3p and its downstream target Gdf-5 regulate social fear expression and potentially mediate oxytocin-induced reversal of social fear.

Detection, processing and reinforcement of social cues: regulation by the oxytocin system.

Many social behaviours are evolutionarily conserved and are essential for the healthy development of an individual. The neuropeptide oxytocin (OXT) is crucial for the fine-tuned regulation of social interactions in mammals. The advent and application of state-of-the-art methodological approaches that allow the activity of neuronal circuits involving OXT to be monitored and functionally manipulated in laboratory mammals have deepened our understanding of the roles of OXT in these behaviours. In this Review, we discuss how OXT promotes the sensory detection and evaluation of social cues, the subsequent approach and display of social behaviour, and the rewarding consequences of social interactions in selected reproductive and non-reproductive social behaviours. Social stressors - such as social isolation, exposure to social defeat or social trauma, and partner loss - are often paralleled by maladaptations of the OXT system, and restoring OXT system functioning can reinstate socio-emotional allostasis. Thus, the OXT system acts as a dynamic mediator of appropriate behavioural adaptations to environmental challenges by enhancing and reinforcing social salience and buffering social stress.

Oxytocin Receptor Expression in Hair Follicle Stem Cells: A Promising Model for Biological and Therapeutic Discovery in Neuropsychiatric Disorders.

The intricate nature of the human brain and the limitations of existing model systems to study molecular and cellular causes of neuropsychiatric disorders represent a major challenge for basic research. The promising progress in patient-derived stem cell technology and in our knowledge on the role of the brain oxytocin (OXT) system in health and disease offer new possibilities in that direction. In this study, the rat hair follicle stem cells (HFSCs) were isolated and expanded in vitro. The expression of oxytocin receptors (OXTR) was evaluated in these cells. The cellular viability was assessed 12 h post stimulation with OXT. The activation of OXTR-coupled intracellular signaling cascades, following OXT treatment was determined. Also, the influence of OXT on neurite outgrowth and cytoskeletal rearrangement were defined. The assessment of OXTR protein expression revealed this receptor is expressed abundantly in HFSCs. As evidenced by the cell viability assay, no adverse or cytotoxic effects were detected following 12 h treatment with different concentrations of OXT. Moreover, OXTR stimulation by OXT resulted in ERK1/2, CREB, and eEF2 activation, neurite length alterations, and cytoskeletal rearrangements that reveal the functionality of this receptor in HFSCs. Here, we introduced the rat HFSCs as an easy-to-obtain stem cell model that express functional OXTR. This cell-based model can contribute to our understanding of the progression and treatment of neuropsychiatric disorders with oxytocinergic system deficiency.

Selective breeding of rats for high (HAB) and low (LAB) anxiety-related behaviour: A unique model for comorbid depression and social dysfunctions.

Animal models of selective breeding for extremes in emotionality are a strong experimental approach to model psychopathologies. They became indispensable in order to increase our understanding of neurobiological, genetic, epigenetic, hormonal, and environmental mechanisms contributing to anxiety disorders and their association with depressive symptoms or social deficits. In the present review, we extensively discuss Wistar rats selectively bred for high (HAB) and low (LAB) anxiety-related behaviour on the elevated plus-maze. After 30 years of breeding, we can confirm the prominent differences between HAB and LAB rats in trait anxiety, which are accompanied by consistent differences in depressive-like, social and cognitive behaviours. We can further confirm a single nucleotide polymorphism in the vasopressin promotor of HAB rats causative for neuropeptide overexpression, and show that low (or high) anxiety and fear levels are unlikely due to visual dysfunctions. Thus, HAB and LAB rats continue to exist as a reliable tool to study the multiple facets underlying the pathology of high trait anxiety and its comorbidity with depression-like behaviour and social dysfunctions.

HDAC1-mediated regulation of GABA signaling within the lateral septum facilitates long-lasting social fear extinction in male mice.

Social anxiety disorder (SAD) is caused by traumatic social experiences. It is characterized by intense fear and avoidance of social contexts, which can be robustly mimicked by the social fear conditioning (SFC) paradigm. The extinction phase of the SFC paradigm is akin to exposure therapy for SAD and requires learning to disassociate the trauma with the social context. Learning-induced acetylation of histones is critical for extinction memory formation and its endurance. Although class I histone deacetylases (HDACs) regulate the abovementioned learning process, there is a lack of clarity in isoforms and spatial specificity in HDAC function in social learning. Utilizing the SFC paradigm, we functionally characterized the role of HDAC1, specifically in the lateral septum (LS), in regulating the formation of long-term social fear extinction memory. We measured a local increase in activity-inducing HDAC1 phosphorylation at serine residues of social fear-conditioned (SFC+) mice in response to the extinction of social fear. We also found that LS-HDAC1 function negatively correlates with acute social fear extinction learning using pharmacological and viral approaches. Further, inhibition of LS-HDAC1 enhanced the expression of the GABA-A receptor ß1 subunit (Gabrb1) in SFC+ mice, and activation of GABA-A receptors facilitated acute extinction learning. Finally, the facilitation of extinction learning by HDAC1 inhibition or GABA-A receptor activation within the LS led to the formation of long-lasting extinction memory, which persisted even 30 days after extinction. Our results show that HDAC1-mediated regulation of GABA signaling in the LS is crucial for the formation of long-lasting social fear extinction memory.

Advances in human oxytocin measurement: challenges and proposed solutions.

Oxytocin, a neuropeptide known for its role in reproduction and socioemotional processes, may hold promise as a therapeutic agent in treating social impairments in patient populations. However, research has yet to uncover precisely how to manipulate this system for clinical benefit. Moreover, inconsistent use of standardized and validated oxytocin measurement methodologies-including the design and study of hormone secretion and biochemical assays-present unresolved challenges. Human studies measuring peripheral (i.e., in plasma, saliva, or urine) or central (i.e., in cerebrospinal fluid) oxytocin concentrations have involved very diverse methods, including the use of different assay techniques, further compounding this problem. In the present review, we describe the scientific value in measuring human endogenous oxytocin concentrations, common issues in biochemical analysis and study design that researchers face when doing so, and our recommendations for improving studies using valid and reliable methodologies.

Synthetic Oxytocin and Vasopressin Act Within the Central Amygdala to Exacerbate Aggression in Female Wistar Rats.

Exacerbated aggression is a high-impact, but poorly understood core symptom of several psychiatric disorders, which can also affect women. Animal models have successfully been employed to unravel the neurobiology of aggression. However, despite increasing evidence for sex-specificity, little is known about aggression in females. Here, we studied the role of the oxytocin (OXT) and arginine vasopressin (AVP) systems within the central amygdala (CeA) on aggressive behavior displayed by virgin female Wistar rats using immunohistochemistry, receptor autoradiography, and neuropharmacology. Our data show that CeA GABAergic neurons are activated after an aggressive encounter in the female intruder test. Additionally, neuronal activity (pERK) negatively correlated with the display of aggression in low-aggressive group-housed females. Binding of OXT receptors, but not AVP-V1a receptors, was increased in the CeA of high-aggressive isolated and trained (IST) females. Finally, local infusion of either synthetic OXT or AVP enhanced aggression in IST females, whereas blockade of either of these receptors did not affect aggressive behavior. Altogether, our data support a moderate role of the CeA in female aggression. Regarding neuropeptide signaling, our findings suggest that synthetic, but not endogenous OXT and AVP modulate aggressive behavior in female Wistar rats.

A model for the social neuroscience of music production begins on a dubious note: Commentary on Greenberg et al. (2021).

Group singing and music-making behaviors that were rapidly adapted to the coronavirus disease (COVID-19) pandemic context suggest to Greenberg et al. (2021) not only a musical solution to pandemic-related social isolation but also the importance of the social neuroscientific side of music. They propose a model of the social neuroscience of music production premised on the view that group singing leads to increased levels of oxytocin (a neuropeptide associated with empathy and social bonding), citing data of Schladt et al. (2017) and Keeler et al. (2015) as support. The present commentary points out that Schladt et al. reported a decrease rather than an increase in oxytocin level following group singing. Further, reference to the work by Keeler et al. (2015) is only partially accurate, and evidence contrary to the oxytocin premise is ignored. Similar inaccuracy is associated with claims for cortisol, another primary component of their model. While the authors are applauded for directing attention to both the social neuroscience of music and the value of group singing, tempering the stated premises associated with the oxytocin and cortisol channels of the model is recommended. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Modelling sexual violence in male rats: the sexual aggression test (SxAT).

Sexual assault and rape are crimes that impact victims worldwide. Although the psychosocial and eco-evolutionary factors associated with this antisocial behavior have repeatedly been studied, the underlying neurobiological mechanisms are still largely unknown. Here, we established a novel paradigm to provoke and subsequently assess sexual aggression (SxA) in adult male Wistar rats: the sexual aggression test (SxAT). Briefly, male Wistar rats are sexually aroused by a receptive female, which is exchanged by a non-receptive female immediately after the first intromission. This protocol elicits forced mounting and aggressive behavior toward the non-receptive female to different degrees, which can be scored. In a series of experiments we have shown that SxA behavior is a relatively stable trait in rats and correlates positively with sexual motivation. Rats with innate abnormal anxiety and aggressive behavior also show abnormal SxA behavior. In addition, central infusion of oxytocin moderately inhibits aggressive behavior, but increases forced mounting. Finally, we identified the agranular insular cortex to be specifically activated by SxA, however, inhibition of this region did not significantly alter behavior in the SxAT. Altogether, the SxAT is a paradigm that can be readily implemented in behavioral laboratories as a valuable tool to find answers regarding the biological mechanisms underlying SxA in humans, as well as social decision-making in general.

Translocator protein (18kDa) TSPO: a new diagnostic or therapeutic target for stress-related disorders?

Efficient treatment of stress-related disorders, such as depression, is still a major challenge. The onset of antidepressant drug action is generally quite slow, while the anxiolytic action of benzodiazepines is considerably faster. However, their long-term use is impaired by tolerance development, abuse liability and cognitive impairment. Benzodiazepines act as positive allosteric modulators of É£-aminobutyric acid type A (GABAA) receptors. 3α-reduced neurosteroids such as allopregnanolone also are positive allosteric GABAA receptor modulators, however, through a site different from that targeted by benzodiazepines. Recently, the administration of neurosteroids such as brexanolone or zuranolone has been shown to rapidly ameliorate symptoms in post-partum depression or major depressive disorder. An attractive alternative to the administration of exogenous neurosteroids is promoting endogenous neurosteroidogenesis via the translocator protein 18k Da (TSPO). TSPO is a transmembrane protein located primarily in mitochondria, which mediates numerous biological functions, e.g., steroidogenesis and mitochondrial bioenergetics. TSPO ligands have been used in positron emission tomography (PET) studies as putative markers of microglia activation and neuroinflammation in stress-related disorders. Moreover, TSPO ligands have been shown to modulate neuroplasticity and to elicit antidepressant and anxiolytic therapeutic effects in animals and humans. As such, TSPO may open new avenues for understanding the pathophysiology of stress-related disorders and for the development of novel treatment options.

Transcriptome and chromatin alterations in social fear indicate association of MEG3 with successful extinction of fear.

Social anxiety disorder is characterized by a persistent fear and avoidance of social situations, but available treatment options are rather unspecific. Using an established mouse social fear conditioning (SFC) paradigm, we profiled gene expression and chromatin alterations after the acquisition and extinction of social fear within the septum, a brain region important for social fear and social behaviors. Here, we particularly focused on the successful versus unsuccessful outcome of social fear extinction training, which corresponds to treatment responsive versus resistant patients in the clinics. Validation of coding and non-coding RNAs revealed specific isoforms of the long non-coding RNA (lncRNA) Meg3 regulated, depending on the success of social fear extinction. Moreover, PI3K/AKT was differentially activated with extinction success in SFC-mice. In vivo knockdown of specific Meg3 isoforms increased baseline activity of PI3K/AKT signaling, and mildly delayed social fear extinction. Using ATAC-Seq and CUT&RUN, we found alterations in the chromatin structure of specific genes, which might be direct targets of lncRNA Meg3.

Neuropeptide Y, calcitonin gene-related peptide, and neurokinin A in brain regions of HAB rats correlate with anxiety-like behaviours.

Anxiety disorders are pervasive psychiatric disorders causing great suffering. The high (HAB) and low (LAB) anxiety-related behaviour rats were selectively bred to investigate neurobiological correlates of anxiety. We compared the level of neuropeptides relevant for anxiety- and depression-related behaviours in selected brain regions of HAB and LAB rats. Increased anxiety and depression-like behaviours of male and female HAB rats in the elevated plus-maze and forced swim tests were accompanied by elevated levels of neuropeptide Y (NPY) in the prefrontal (PFC), frontal (FC) and cingulate cortex (CCx), the striatum, and periaqueductal grey (PAG). Moreover, HAB rats displayed sex-dependent, elevated levels of calcitonin gene-related peptide (CGRP) in PFC, FC, CCx, hippocampus, and PAG. Higher neurokinin A (NKA) levels were detected in CCx, striatum, and PAG in HAB males and in CCx and hypothalamus in HAB females. Increased neurotensin was detected in CCx and PAG in HAB males and in hypothalamus in HAB females. Elevated corticotropin-releasing hormone (CRH) levels appeared in female HAB hypothalamus. Significant correlations were found between anxiety-like behaviour and NPY, CGRP, NKA, and neurotensin, particularly with NPY in CCx and striatum, CGRP in FC and hippocampus, and NKA in entorhinal cortex. This is the first report of NPY, CGRP, NKA, Neurotensin, and CRH measurements in brain regions of HAB and LAB rats, which showed widespread NPY and CGRP alterations in cortical regions, with NKA and neurotensin changes localised in sub-cortical areas. The results may contribute to elucidate pathophysiological mechanisms underlying anxiety and depression and should facilitate identifying novel therapeutic targets.

Structure-function relationships of the disease-linked A218T oxytocin receptor variant.

Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.

Neurobiology of the lateral septum: regulation of social behavior.

Social interactions are essential for mammalian life and are regulated by evolutionary conserved neuronal mechanisms. An individual's internal state, experiences, and the nature of the social stimulus are critical for determining apt responses to social situations. The lateral septum (LS) - a structure of the basal forebrain - integrates abundant cortical and subcortical inputs, and projects to multiple downstream regions to generate appropriate behavioral responses. Although incoming cognitive information is indispensable for contextualizing a social stimulus, neuromodulatory information related to the internal state of the organism significantly influences the behavioral outcome as well. This review article provides an overview of the neuroanatomical properties of the LS, and examines its neurochemical (neuropeptidergic and hormonal) signaling, which provide the neuromodulatory information essential for fine-tuning social behavior across the lifespan.

Consequences of pandemic-associated social restrictions: Role of social support and the oxytocin system.

During pandemics, governments take drastic actions to prevent the spreading of the disease, as seen during the present COVID-19 crisis. Sanctions of lockdown, social distancing and quarantine urge people to exclusively work and teach at home and to restrict social contacts to a minimum; lonely people get into further isolation, while families` nerves are strained to the extreme. Overall, this results in a dramatic and chronic increase in the level of psychosocial stress over several months mainly caused by i) social isolation and ii) psychosocial stress associated with overcrowding, social tension in families, and domestic violence. Moreover, pandemic-associated social restrictions are accompanied by loss of an essential stress buffer and important parameter for general mental and physical health: social support. Chronic psychosocial stress and, in particular, social isolation and lack of social support affect not only mental health, but also the brain oxytocin system and the immune system. Hence, pandemic-associated social restrictions are expected to increase the risk of developing psychopathologies, such as depression, anxiety-related and posttraumatic stress disorders, on the one hand, but also to induce a general inflammatory state and to impair the course of infectious disorders on the other. Due to its pro-social and stress-buffering effects, resulting in an anti-inflammatory state in case of disease, the role of the neuropeptide oxytocin will be discussed and critically considered as an emerging treatment option in cases of pandemic-induced psychosocial stress, viral infection and during recovery. In this review, we aim to critically focus on possible short- and long-term consequences of social restrictions on mental health and the immune system, while discussion oxytocin as a possible treatment option.

Co-Stimulation of Oxytocin and Arginine-Vasopressin Receptors Affect Hypothalamic Neurospheroid Size.

Oxytocin (OXT) is a neuropeptide involved in a plethora of behavioral and physiological processes. However, there is a prominent lack of 3D cell culture models that investigate the effects of OXT on a cellular/molecular level. In this study, we established a hypothalamic neuronal spheroid model to investigate the cellular response in a more realistic 3D setting. Our data indicate that the formation of spheroids itself does not alter the basic characteristics of the cell line and that markers of cellular morphology and connectivity are stably expressed. We found that both OXT and arginine vasopressin (AVP) treatment increase spheroid size (surface area and volume), as well as individual nucleus size, which serves as an indicator for cellular proliferation. The cellular response to both OXT and AVP seems mainly to be mediated by the AVP receptor 1a (V1aR); however, the OXT receptor (OXTR) contributes significantly to the observed proliferative effect. When we blocked the OXTR pharmacologically or knocked down the OXTR by siRNA, the OXT- or AVP-induced cellular proliferation decreased. In summary, we established a 3D cell culture model of the neuronal response to OXT and AVP and found that spheroids react to the treatment via their respective receptors but also via cross-talk between the two receptor types.

Chronic oxytocin-driven alternative splicing of Crfr2α induces anxiety.

The neuropeptide oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including anxiety and autism spectrum disorders. However, the behavioral and molecular consequences associated with chronic OXT treatment and chronic receptor (OXTR) activation have scarcely been studied, despite the potential therapeutic long-term use of intranasal OXT. Here, we reveal that chronic OXT treatment over two weeks increased anxiety-like behavior in rats, with higher sensitivity in females, contrasting the well-known anxiolytic effect of acute OXT. The increase in anxiety was transient and waned 5 days after the infusion has ended. The behavioral effects of chronic OXT were paralleled by activation of an intracellular signaling pathway, which ultimately led to alternative splicing of hypothalamic corticotropin-releasing factor receptor 2α (Crfr2α), an important modulator of anxiety. In detail, chronic OXT shifted the splicing ratio from the anxiolytic membrane-bound (mCRFR2α) form of CRFR2α towards the soluble CRFR2α (sCRFR2α) form. Experimental induction of alternative splicing mimicked the anxiogenic effects of chronic OXT, while sCRFR2α-knock down reduced anxiety-related behavior of male rats. Furthermore, chronic OXT treatment triggered the release of sCRFR2α into the cerebrospinal fluid with sCRFR2α levels positively correlating with anxiety-like behavior. In summary, we revealed that the shifted splicing ratio towards expression of the anxiogenic sCRFR2α underlies the adverse effects of chronic OXT treatment on anxiety.