Human Oxytocin Research Articles

Sex Differences in the Hypothalamic Oxytocin Pathway to Locus Coeruleus and Augmented Attention with Chemogenetic Activation of Hypothalamic Oxytocin Neurons.

The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are well recognized as essential for focused arousal and novelty-oriented responses, while many children with autism spectrum disorder (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli. This has led to the hypothesis that atypical LC activity may be involved in ASD. Oxytocin (OXT) neurons and receptors are known to play an important role in social behavior, pair bonding and cognitive processes and are under investigation as a potential treatment for ASD. However, little is known about the neurotransmission from hypothalamic paraventricular (PVN) OXT neurons to LC NA neurons. In this study, we test, in male and female rats, whether PVN OXT neurons excite LC neurons, whether oxytocin is released and involved in this neurotransmission, and whether activation of PVN OXT neurons alters novel object recognition. Using “oxytocin sniffer cells” (CHO cells that express the human oxytocin receptor and a Ca indicator) we show that there is release of OXT from hypothalamic PVN OXT fibers in the LC. Optogenetic excitation of PVN OXT fibers excites LC NA neurons by co-release of OXT and glutamate, and this neurotransmission is greater in males than females. In male, but not in female animals, chemogenetic activation of PVN OXT neurons increases attention to novel objects.

Oxytocin-Trust Link in Oxytocin-Sensitive Participants and Those Without Autistic Traits.

There have been numerous studies in which the biological role of oxytocin in trusting behavior has been investigated. However, a link between oxytocin and trust in humans was discovered only in one early study. We hypothesized that there is a large interindividual variation in oxytocin sensitivity, and that such variation is one reason for the doubt surrounding the role of oxytocin in trusting behavior. Here, in a double-blind, prospective, case-control study, we administered intranasal oxytocin to participants of trust and risk games. We measured salivary oxytocin concentration, relating it to the amount of money transferred among participants (a proxy for trust) and the autism-spectrum quotient (AQ). A one-sided Fisher’s exact test was performed to detect differences between the oxytocin and placebo groups in the proportions of investors who transferred the maximum amount of money. We discovered a tendency for participants who received oxytocin to transfer higher amounts of money to co-participants than those who received a placebo (P = 0.04). We also revealed a high degree of interindividual variation in salivary oxytocin concentrations after oxytocin administration. After stratifying the samples with respect to oxytocin sensitivity, oxytocin-sensitive participants in the oxytocin group also transferred higher amounts of money than those in the placebo group (P = 0.03), while such a tendency was not observed for oxytocin-insensitive participants (P = 0.34). Participants with lower AQ scores (less severe autistic traits) exhibited a greater tendency toward trusting behavior after oxytocin administration than did those with higher AQ scores (P = 0.02). A two-sample t-test that was performed to detect significant differences in the mean transfers between the oxytocin and placebo groups indicated no significant between-group difference in the mean transfers (P = 0.08). There are two possible interpretations of these results: First, there is no effect of oxytocin on trust in humans; second, the effects of oxytocin on trust in humans is person-dependent. However, the results should be interpreted with caution as the effect size was not larger than the minimal detectable effect size and the results were not statistically significant (P > 0.05) after Bonferroni corrections.

Oxytocinergic Modulation of Threat-Specific Amygdala Sensitization in Humans Is Critically Mediated by Serotonergic Mechanisms

BackgroundOverarching conceptualizations propose that the complex social-emotional effects of oxytocin (OXT) in humans are partly mediated by interactions with other neurotransmitter systems. Recent animal models suggest that the anxiolytic effects of OXT are critically mediated by the serotonin (5-HT) system, yet direct evidence in humans is lacking. MethodsTo determine the role of 5-HT in OXT-induced attenuation of amygdala threat reactivity and sensitization/desensitization, we conducted a parallel-group, randomized, placebo-controlled, double-blind experiment during which 121 healthy subjects underwent a transient decrease in 5-HT signaling via acute tryptophan depletion or the corresponding placebo-control protocol before the administration of intranasal OXT or placebo intranasal spray, respectively. Mean and repetition-dependent changes in threat-specific amygdala reactivity toward threatening stimuli (angry faces) as assessed by functional magnetic resonance imaging served as the primary outcome. ResultsNo main or interaction effects of treatment on amygdala threat reactivity were observed, yet OXT switched bilateral amygdala threat sensitization to desensitization, and this effect was significantly attenuated during decreased central 5-HT signaling via pretreatment with acute tryptophan depletion. ConclusionsThe present findings provide the first evidence for a role of OXT in threat-specific amygdala desensitization in humans and suggest that these effects are critically mediated by the 5-HT system. OXT may have a therapeutic potential to facilitate amygdala desensitization, and adjunct upregulation of 5-HT neurotransmission may facilitate OXT’s anxiolytic potential.

Directed evolution for high functional production and stability of a challenging G protein-coupled receptor

Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.

Validation of a commercial enzyme immunoassay to assess urinary oxytocin in humans.

Within the last decade, oxytocin (OT) has attracted a lot of attention in the context of various human social behaviors. Besides its importance in regulating physiological processes in females related to giving birth and lactation, OT is involved in the establishment and maintenance of social relationships, trust and emotion recognition. However, results are not always consistent across studies, which may partly be due to the incomplete validation of methods used to assess OT levels. Carefully validating a method before its use is of crucial importance to ensure that it can be used to accurately, reliably and repeatedly assess OT levels. With this study we evaluated a commercially available Enzyme Immunoassay to assess OT in human urine samples by conducting a careful analytical validation. Results indicate that, with regard to parallelism and immunoreactivity, human urinary OT can be assessed reliably. However, extraction methods need further improvement to optimize measures of accuracy and extraction efficiency, especially in the lower range of the assay system. Tests on OT stability indicate that OT is affected by degradation when stored at 4°C or room temperature. Storing urine samples over longer periods revealed that OT levels are most stable when stored as ethanol extracts at −20°C compared to being stored as samples at −20°C or −80°C. Although some of the validated parameters did not reach the intended quality criteria, this study highlights the importance of such in depth validation procedures and reporting results to make them available to researchers embarking on projects utilizing such methods.

Chapter 6 - Oxytocin, eating behavior, and metabolism in humans

The hypothalamic peptide oxytocin has been increasingly recognized as a hormone and neurotransmitter with important effects on energy intake, metabolism, and body weight and is under investigation as a potential novel therapeutic agent for obesity. The main neurons producing oxytocin and expressing the oxytocin receptor are strategically located in brain areas known to be critically involved in homeostatic energy balance as well as hedonic and motivational aspects of eating behavior. In this chapter, we will review the central and peripheral physiology of oxytocin and the interaction of oxytocin with key hormones and neural circuitries that affect food intake and metabolism. Next, we will synthesize the available data on endogenous oxytocin levels related to caloric intake, body weight, and metabolic status. We will then review the effects of exogenous oxytocin administration on eating behavior, body weight, and metabolism in humans, including in healthy individuals as well as specific populations with suspected perturbations involving oxytocin pathways. Finally, we will address the promise and fundamental challenges of translating this line of research to clinical care.

Oxytocin Administration Increases Proactive Control in Men with Overweight or Obesity: A Randomized, Double-Blind, Placebo-Controlled Crossover Study.

Preclinical and clinical evidence suggests that oxytocin administration decreases food intake and weight. The mechanisms underlying the anorexigenic effects of oxytocin in humans are unknown but critical to study to consider oxytocin as a neurohormonal weight loss treatment. Complementing ongoing research into metabolic and food motivation mechanisms of oxytocin, this study hypothesized that in humans, oxytocin improves cognitive control over behavior. In a randomized, double-blind, placebo-controlled crossover study of 24-IU single-dose intranasal oxytocin, 10 men with overweight or obesity completed a stop-signal task assessing ability and strategy to suppress behavioral impulses, in which they performed a choice-reaction task (go task) but had to withhold their response when prompted (stop task). It was hypothesized that oxytocin would improve suppression of behavioral impulses. After receiving oxytocin, compared with placebo, participants showed increased reaction times in the go task (mean [M] = 936 milliseconds vs. 833 millseconds; P = 0.012; 95% CI: 29 to 178) and displayed fewer stop errors (M = 36.41% vs. 41.15%; P = 0.049; 95% CI: -9.43% to -0.03%). Oxytocin triggers increased proactive control over behavior. Future studies need to further characterize the impact of oxytocin on cognitive control and investigate its potential role in the anorexigenic effects of oxytocin in human obesity.

Links Between the Neurobiology of Oxytocin and Human Musicality

The human species possesses two complementary, yet distinct, universal communication systems—language and music. Functional imaging studies have revealed that some core elements of these two systems are processed in closely related brain regions, but there are also clear differences in brain circuitry that likely underlie differences in functionality. Music affects many aspects of human behavior, especially in encouraging prosocial interactions and promoting trust and cooperation within groups of culturally compatible but not necessarily genetically related individuals. Music, presumably via its impact on the limbic system, is also rewarding and motivating, and music can facilitate aspects of learning and memory. In this review these special characteristics of music are considered in light of recent research on the neuroscience of the peptide oxytocin, a hormone that has both peripheral and central actions, that plays a role in many complex human behaviors, and whose expression has recently been reported to be affected by music-related activities. I will first briefly discuss what is currently known about the peptide’s physiological actions on neurons and its interactions with other neuromodulator systems, then summarize recent advances in our knowledge of the distribution of oxytocin and its receptor (OXTR) in the human brain. Next, the complex links between oxytocin and various social behaviors in humans are considered. First, how endogenous oxytocin levels relate to individual personality traits, and then how exogenous, intranasal application of oxytocin affects behaviors such as trust, empathy, reciprocity, group conformity, anxiety, and overall social decision making under different environmental conditions. It is argued that many of these characteristics of oxytocin biology closely mirror the diverse effects that music has on human cognition and emotion, providing a link to the important role music has played throughout human evolutionary history and helping to explain why music remains a special prosocial human asset. Finally, it is suggested that there is a potential synergy in combining oxytocin- and music-based strategies to improve general health and aid in the treatment of various neurological dysfunctions.

Crystal structure of the human oxytocin receptor.

The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein-coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.

Oxitozina erabilgarria izan al daiteke autismoan gertatzen den urritasun sozialerako?

Autismoaren espektroaren nahastea jokabidean definitutako garapen neurologikoaren desoreka da. Bi sintoma dira haren bereizgarri nagusi: batetik, urritasun soziala; bestetik, mugimendu errepikakor eta murriztaileak. Autismoaren klinikaren heterogeneotasunagatik, eta etiologia eta fisiopatologia konplexuagatik, farmakoterapia zuzendu bat garatzea da gaur egungo erronka garrantzitsua. Erabiltzen den ohiko interbentzioa portaeraren terapia bada ere, autismo kasuen % 75ek tratamendu farmakologikoa jasotzen du asoziaturiko sintometarako, baina momentuz ez dago sintoma nagusietarako eraginkorra den farmakorik. Beraz, farmako berriak garatzeko ahalegin handia egiten ari da ikerketa biomedikoa, eta horien artean oxitozina dugu. oxitozina nerbio sistema zentralean neuromodulatzaile gisa jokatzen duen peptido bat da. Ikusi da garapen goiztiarrean mutazio genetikoengatik gertatutako oxitozina-sistemaren porrotak jokaera sozialean eragin dezakeela eta aktibitate eta plastikotasun sinaptikoan ere bai. testuinguru horretan, oxitozinak autismoaren tratamenduan izan dezakeen erabilgarritasuna eta arrakasta aztertzen ari dira. Batetik, autismoaren forma monogenikoetan oinarritutako zenbait animalia-ereduk oxitozina sisteman nolabaiteko alterazioak erakusten dituzte, eta horietan oxitozina emateak urritasun sozialaren hobekuntza eragiten du. Bestetik, gizakietan ere hainbat saio kliniko egiten ari dira oxitozinaren erabilgarritasun terapeutikoa aztertzeko. Baina kasu horretan, animalia-ereduetan ikusi denarekin konparaturik, lortutako emaitzak ezberdinak dira: ikerketa batzuetan kognizio sozialean hobekuntzak ikusi dira, baina beste batzuetan ez. Gizakien artean dagoen heterogeneotasuna dela eta, ez da oso harritzekoa hori. ondorioz, ikerketa gehiagoren beharra dago oxitozinak autismoan izan dezakeen erabilgarritasuna modu sendoago batean balioztatzeko eta baita tratamendu mota hau zein banakorentzat litzatekeen onuragarriena zehazteko ere.

Comparison of the pharmacologic profiles of arginine vasopressin and oxytocin analogs at marmoset, titi monkey, macaque, and human oxytocin receptors

The oxytocin-arginine vasopressin (OT-AVP) ligand-receptor family influences a variety of physiological, behavioral, and social behavioral processes in the brain and periphery. The OT-AVP family is highly conserved in mammals, but recent discoveries have revealed remarkable diversity in OT ligands and receptors in New World Monkeys (NWMs) providing a unique opportunity to assess the effects of genetic variation on pharmacological signatures of peptide ligands. The consensus mammalian OT sequence has leucine in the 8th position (Leu8-OT), whereas a number of NWMs, including the marmoset, have proline in the 8th position (Pro8-OT) resulting in a more rigid tail structure. OT and AVP bind to OT’s cognate G-protein coupled receptor (OTR), which couples to various G-proteins (Gi/o, Gq, Gs) to stimulate diverse signaling pathways. CHO cells expressing marmoset (mOTR), titi monkey (tOTR), macaque (qOTR), or human (hOTR) OT receptors were used to compare AVP and OT analog-induced signaling. Assessment of Gq-mediated increase in intracellular calcium (Ca2+) demonstrated that AVP was less potent than OT analogs at OTRs from species whose endogenous ligand is Leu8-OT (tOTR, qOTR, hOTR), relative to Pro8-OT. Likewise, AVP-induced membrane hyperpolarization was less potent at these same OTRs. Evaluation of (Ca2+)-activated potassium (K+) channels using the inhibitors apamin, paxilline, and TRAM-34 demonstrated that both intermediate and large conductance Ca2+-activated K+ channels contributed to membrane hyperpolarization, with different pharmacological profiles identified for distinct ligand-receptor combinations. Understanding more fully the contributions of structure activity relationships for these peptide ligands at vasopressin and OT receptors will help guide the development of OT-mediated therapeutics.

Null results of oxytocin and vasopressin administration across a range of social cognitive and behavioral paradigms: Evidence from a randomized controlled trial

Research examining oxytocin and vasopressin in humans has the potential to elucidate neurobiological mechanisms underlying human sociality that have been previously unknown or not well characterized. A primary goal of this work is to increase our knowledge about neurodevelopmental and psychiatric disorders characterized by impairments in social cognition. However, years of research highlighting wide-ranging effects of, in particular, intranasal oxytocin administration have been tempered as the fields of psychology, neuroscience, and other disciplines have been addressing concerns over the reproducibility and validity of research findings. We present a series of behavioral tasks that were conducted using a randomized, double-blind, placebo controlled, between-subjects design, in which our research group found no main effects of oxytocin and vasopressin on a host of social outcomes. In addition to null hypothesis significance testing, we implemented equivalence testing and Bayesian hypothesis testing to examine the sensitivity of our findings. These analyses indicated that 47–83% of our results (depending on the method of post-hoc analysis) had enough sensitivity to detect the absence of a main effect. Our results add to evidence that intranasal oxytocin may have a more limited direct effect on human social processes than initially assumed and suggest that the direct effects of intranasal vasopressin may be similarly limited. Randomized controlled trial registration: NCT01680718.

Discovery of Potent, Selective, and Short-Acting Peptidic V2 Receptor Agonists.

The vasopressin analogue desmopressin (desamino-d-arginine8 vasopressin, dDAVP, 1) is a potent vasopressin 2 (V2) receptor (V2R) agonist approved in many countries for the treatment of diabetes insipidus, primary nocturnal enuresis, nocturia, and coagulation disorders. Since 1 is primarily excreted via the kidneys, an age-related decline in kidney function leads to slower elimination, prolonged antidiuresis, and hyponatremia. In search of novel, potent, selective, and short-acting peptidic V2R agonists, we synthesized a series of C-terminally truncated analogues of [Val4]dDAVP, 2, modified in positions 2, 3, and 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency at the human V2 receptor, selectivity versus the related receptors (human vasopressin 1a receptor, human vasopressin 1b receptor, and human oxytocin receptor), and pharmacokinetic profiles in rodents and other higher species. The truncated analogues show excellent potency at the V2R, increased systemic clearance, and shorter half-life in rats. Two compounds 19 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-Agm) and 38 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-d-Arg-NEt2) have been selected for clinical development for nocturia.

Oxytocin Selectively Improves Empathic Accuracy: A Replication in Men and Novel Insights in Women

BackgroundPreviously, oxytocin, a neuropeptide implicated in human social cognition and behavior, was shown to improve people’s ability to dynamically track another’s emotional state (“empathic accuracy”) specifically for less socially proficient individuals—i.e., healthy adults who score higher on the Autism Spectrum Quotient (AQ); conversely, oxytocin had no effect on empathic accuracy for more socially proficient individuals, who performed well following oxytocin and placebo. Here, we aimed to replicate this finding and investigate the effects of oxytocin on empathic accuracy in women. To date, women have been seriously underrepresented in human oxytocin research, and it is not known whether the effects observed in male-only samples apply to women. MethodsIn this randomized, double-blind, placebo-controlled, crossover trial, we administered 24 IU intranasal oxytocin (and, on a separate occasion, a matching placebo) to 31 men and 40 women and then measured empathic accuracy. AQ was assessed at baseline (prior to drug administration). ResultsReplicating a 2010 study by Bartz et al., oxytocin selectively improved empathic accuracy for men who scored higher on the AQ, whereas oxytocin did not benefit their lower AQ counterparts. Conversely, we found no effect of oxytocin on empathic accuracy for women (regardless of their AQ score). ConclusionsIn addition to speaking to reliability, this research is important given interest in using oxytocin to augment social functioning in some psychiatric disorders marked by social cognitive impairments. More generally, this research adds to our understanding of the biological systems that support human sociality and provides further evidence for the role of oxytocin therein.

A Comparison of the Ability of Leu8- and Pro8-Oxytocin to Regulate Intracellular Ca2+ and Ca2+-Activated K+ Channels at Human and Marmoset Oxytocin Receptors.

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.

Cligosiban, A Novel Brain-Penetrant, Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents

IntroductionFew treatments are available for men with premature ejaculation (PE); oxytocin (OT) receptor antagonism in the central nervous system (CNS) is a potential new approach. AimTo determine if cligosiban selectively inhibits human OT receptors, penetrates the CNS, shows pharmacology in the CNS, and effects ejaculatory physiology in pre-clinical systems. MethodsExperiments complied with United Kingdom legislation and were subject to local ethical review. In vitro potency and selectivity of cligosiban was assessed using recombinant and native OT receptor systems including both neuronal and non-neuronal cell types. Selectivity was determined over neighboring V1A, V1B, and V2 vasopressin receptors using a combination of recombinant and native vasopressin receptor assay systems. To determine an effect on central OT receptors and on ejaculation, cligosiban was evaluated in 2 anesthetized rat models—the electromyography model of ejaculatory physiology and a model of OT-mediated CNS neuronal firing. The CNS penetration of cligosiban was also determined by measuring cerebrospinal fluid and plasma drug concentrations following an intravenous (IV) infusion in rats. Main Outcome MeasureThese were functional measures of pharmacology in vitro, in cell lines and tissues, and in vivo in rats. ResultsCligosiban is a potent OT receptor antagonist, with a base dissociation constant of 5.7 nmol/L against native human uterine smooth muscle cell OT receptors. Cligosiban displays similar antagonistic potency against human recombinant and rat native OT receptors, including neuronal OT receptors. Cligosiban demonstrates >100-fold selectivity over human V1A, V1B, and V2 vasopressin receptors. In the electromyography model, cligosiban (0.9 mg/kg, IV bolus) reduced the bulbospongiosum burst pattern and contraction amplitude associated with ejaculation. In the anesthetized CNS neuronal firing model, the same dosing regimen of cligosiban (0.9 mg/kg IV bolus) modulated the OT-mediated response in the nucleus tractus solitarius. After systemic dosing to rats, cligosiban showed good CNS penetration. Clinical ImplicationsAs the first highly selective and centrally penetrant OT receptor antagonist, cligosiban represents a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE. Strength & LimitationsThe pharmacology and selectivity of cligosiban is determined using functional assays in recombinant cell lines, native cell lines, and tissue. Functional outcomes in in vivo systems are linked to CNS measures of pharmacology. The translation of the animal models of ejaculation to PE in man is unproven. ConclusionCligosiban, a potent, selective OT receptor antagonist, demonstrated CNS penetration and pharmacology and, using the same dosing regimen, inhibited apomorphine-induced ejaculation in rats. Cligosiban is a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE.Wayman C, Russell R, Tang K, et al. Cligosiban, A Novel Brain Penetrant Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents. J Sex Med 2018;15:1698–1706.

DNA methylation of OXTR is associated with parasympathetic nervous system activity and amygdala morphology.

Oxytocin has anxiolytic properties whose mechanisms of action are still being identified. DNA methylation in the promoter region of the oxytocin receptor gene (OXTR), an epigenetic modification that putatively reflects a downtuning of the oxytocin system, has previously been implicated in the regulation of fear-related responses through the amygdala. In this study, we attempted to characterize the relationship between methylation of OXTR and anxiogenesis using two distinct endophenotypes: autonomic nervous system activity and subcortical brain structure. In 79 participants, we found that increased OXTR methylation is associated with attenuated resting parasympathetic tone, measured using high-frequency heart rate variability. Further, we found that this relationship is mediated by brain morphology, such that OXTR methylation is associated with increased gray matter of the central amygdala which is, in turn, associated with decreased parasympathetic tone. These results further our understanding of epigenetic regulation of the human oxytocin system and its role in anxiogenesis.

Oxytocin levels in saliva correlate better than plasma levels with concentrations in the cerebrospinal fluid of patients in neurocritical care.

In the converging fields of neuroendocrinology and behavioural neuroscience, the interaction between peripheral secretion and central release of oxytocin in humans has not yet been comprehensively assessed. As the human brain is not directly accessible and as the collection of human cerebrospinal fluid (CSF) usually requires invasive procedures, easier accessible compartments such as blood or saliva attract increasing attention. In this study, we prospectively determined oxytocin concentrations in the three compartments plasma, CSF and saliva of fifty critically ill patients with neurological and neurosurgical diseases. All samples per patient were collected concomitantly. Oxytocin was measured by a highly sensitive and specific radioimmunoassay. Strength of correlation was assessed by the Spearman rank correlation coefficient. Correlation analyses revealed modest to strong correlations for oxytocin between the saliva and CSF compartments while predominantly weak correlations were found between the CSF and plasma as well as between the plasma and saliva compartments. In conclusion, we demonstrated modest to strong correlations between the saliva and CSF compartment suggesting that saliva oxytocin may help to assess CSF oxytocin levels. In contrast, plasma oxytocin failed to correspond well with CSF oxytocin levels as predominantly weak correlations were found between the CSF and plasma as well as between the plasma and saliva compartments which are unlikely to have a biological relevance. Further research is needed to clarify to what extent saliva oxytocin may serve as a biomarker reflecting brain oxytocin activity. This article is protected by copyright. All rights reserved.

Neuropeptide oxytocin enhances μ opioid receptor signaling as a positive allosteric modulator

Oxytocin (OT) is a 9-amine neuropeptide that plays an essential role in mammalian labor, lactation, maternal bonding, and social affiliation. OT has been reported to exert an analgesic effect in both humans and animals, and the results of certain animal experiments have shown that the analgesic effect of OT is partially blocked by opioid receptor antagonists. To investigate the relationship between OT and μ opioid receptor (MOR), we evaluated how OT affects MOR in vitro by performing an electrical impedance-based receptor biosensor assay (CellKey™ assay), an intracellular cAMP assay, and a competitive receptor-binding analysis by using cells stably expressing human MOR and OT receptor. In both the CellKey™ assay and the intracellular cAMP assay, OT alone exerted no direct agonistic effect on human MOR, but treatment with 10−6 M OT markedly enhanced the MOR signaling induced by 10−6 M endomorphin-1, β-endorphin, morphine, fentanyl, and DAMGO. Moreover, in the competitive receptor-binding assay, 10−6 M OT did not alter the affinity of endomorphin-1 or morphine for MOR. These results suggest that OT could function as a positive allosteric modulator that regulates the efficacy of MOR signaling, and thus OT might represent a previously unrecognized candidate analgesic agent.

Basal Serum Oxytocin Levels in Healthy Non‐Pregnant/Lactating Humans are Below 10 pg/mL and Not Detectable by Traditional Orbitrap LCMS

Quantifying peripheral oxytocin (OT) in humans is of increased interest due to its substantial role in cognition and social behavior (e.g. depression, autism). OT is commonly measured by immunoassays, which lack the sensitivity to accurately quantitate the very low basal circulating levels in non‐pregnant/lactating humans due to matrix interferences. This can be resolved by solid phase extraction (SPE) but is inconsistently performed, leading to large variation in OT levels across studies (Leng, 2016). Liquid chromatography mass spectrometry (LCMS) can separate interferences but requires high resolution accurate‐mass (HRAM) capabilities for selective and accurate quantitation.We developed a HRAM orbitrap based LCMS method to measure underivatized native OT from serum after SPE. 0.5 mL serum with OT‐d5 as internal standard underwent C18 SPE followed by Q‐Exactive LCMS analysis with a C18 core‐shell based column (Kinetex, 150 × 3.0 mm; 2.6 μm) and a linear gradient (formic acid/MeCN). The doubly protonated OT ion m/z 562.25503 was selected for MS quantitation due its high signal intensity. Our method LLOQ was 10 pg/mL, which permitted accurate OT measurement in healthy pregnant women (n=4, 16–24 pg/mL), rats (350 pg/mL) and healthy adults after intranasal OT administration. Intra‐day precision was 2–23% and 0% while accuracy was 66~91% and 108~109% at 47.6 and 90.9 pg/mL.A method for ‘total’ OT (OT‐R/A) measuring 500–700 pg/mL in adult plasma was reported recently using LCMSMS after chemical reduction with 1,4‐dithiothreitol, which supposedly disrupted protein binding, followed by alkylation with iodoacetamide to stabilize the reduced product (Brandtzaeg, 2016). Using this method even higher levels were reported from non‐pregnant/lactating subjects (Semba, 2016, Comminos, 2017). However, the masses used for OT‐R/A analysis by Brandtzaeg were for the singly protonated molecule and differed from the theoretical masses by 39.8 ppm while the doubly protonated species used by Semba for quantitation (562.8/839.1(b6); 562.8/286.1(y3); 562.8/446.0(y4) differed by 970 pm. These measured masses were shown in our HRAM LCMS system to be interferences and not OT‐R/A. When we selected a mass window of ≤6 ppm, our HRAM system was unable to detect native OT‐R/A while OT‐R/A was detectable after spiking with OT down to 10 pg/mL plus OT‐d5‐R/A was detected each time after adding OT‐d5 as internal standard.Underivatized OT was correctly measured in monkeys by LCMSMS after SPE purification using MRM transitions 1007‐>723, 285 and 1012‐>723, 290 for the singly protonated native OT and OT‐d5, respectively with basal levels <LLOQ (10 pg/mL; Lee, 2017), which is in excellent agreement with our results. Similarly, others reported mean circulating OT levels <10 pg/mL in non‐pregnant/lactating humans using 2D‐LCMSMS (1–2 pg/mL; Zhang, 2011) with 3 of the 10 subjects <LLOQ (1 pg/mL) and ELISA after SPE (Carson, 2015; mean 6.7 pg/mL) or <2 pg/mL using RIA after SPE (van Londen, 1997; Modahl, 1998). Accurate OT methods find <10 pg/mL OT in serum/plasma of non‐pregnant/lactating/OT medicated humans.PMIDs: Brandtzaeg 27528413; Carson 25349162; Comminos 28112678; Lee 28289281; Leng 27467712; Modahl 9513736; Semba 28508553; van Londen 9326754; Zhang 21609710Support or Funding InformationnoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.