An evolutionary timeline of the oxytocin signaling pathway.

Oxytocin is a neuropeptide associated with both psychological and somatic processes like parturition and social bonding. Although oxytocin homologs have been identified in many species, the evolutionary timeline of the entire oxytocin signaling gene pathway has yet to be described. Using protein sequence similarity searches, microsynteny, and phylostratigraphy, we assigned the genes supporting the oxytocin pathway to different phylostrata based on when we found they likely arose in evolution. We show that the majority (64%) of genes in the pathway are 'modern'. Most of the modern genes evolved around the emergence of vertebrates or jawed vertebrates (540 - 530 million years ago, 'mya'), including OXTR, OXT and CD38. Of those, 45% were under positive selection at some point during vertebrate evolution. We also found that 18% of the genes in the oxytocin pathway are 'ancient', meaning their emergence dates back to cellular organisms and opisthokonta (3500-1100 mya). The remaining genes (18%) that evolved after ancient and before modern genes were classified as 'medium-aged'. Functional analyses revealed that, in humans, medium-aged oxytocin pathway genes are highly expressed in contractile organs, while modern genes in the oxytocin pathway are primarily expressed in the brain and muscle tissue.

Validation of two immunoassays for oxytocin measurements in human saliva.

The objective of this research was to develop and validate two immunoassays for oxytocin measurement in human saliva, one using a monoclonal and the other a polyclonal antibody against oxytocin, whose affinity for oxytocin was tested by an antibody mapping epitope analysis. These assays were analytically validated and used to compare oxytocin concentrations with those obtained with a commercial kit before and after the extraction or reduction/alkylation (R/A) treatments to saliva samples. The assays were also used to evaluate changes in salivary oxytocin concentrations following a physical effort and an induced psychological stress, which have previously been described as situations that cause an increase in salivary oxytocin. Both assays showed to be precise and accurate in the validation studies, and the antibodies used showed a defined binding region in case of the monoclonal antibody, whereas the polyclonal antibody showed binding events through all the oxytocin sequence. Although the monoclonal and polyclonal assays showed a positive correlation, they give results in a different range of magnitude. Both assays showed significant increases in oxytocin concentrations when applied after the physical effort and the psychological stress. This study shows that a variability in the reported values of oxytocin can occur depending on the assay and indicates that the use of different types of antibodies can give a different range of values when measuring oxytocin in saliva.

Oxytocin, GABA, and dopamine interplay in autism.

Oxytocin plays an important role in brain development and is associated with various neurotransmitter systems in the brain. Abnormalities in the production, secretion, and distribution of oxytocin in the brain, at least during some stages of the development, are critical for the pathogenesis of neuropsychiatric diseases, particularly in the autism spectrum disorder. The etiology of autism includes changes in local sensory and dopaminergic areas of the brain, which are also supplied by the hypothalamic sources of oxytocin. It is very important to understand their mutual relationship. In this review, the relationship of oxytocin with several components of the dopaminergic system, gamma-aminobutyric acid (GABA) inhibitory neurotransmission and their alterations in the autism spectrum disorder is discussed. Special attention has been paid to the results describing a reduced expression of inhibitory GABAergic markers in the brain in the context of dopaminergic areas in various models of autism. It is presumed that the altered GABAergic neurotransmission, due to the absence or dysfunction of oxytocin at certain developmental stages, disinhibits the dopaminergic signaling and contributes to the autism symptoms.

Social neuroscience: How we learn to avoid the bully.

During social interactions, individuals evaluate relationships with their peers and switch from approach to avoidance, particularly in response to aggressive encounters. A new study in mice investigated the underlying brain mechanisms and identified oxytocin as a key regulator of social avoidance learning.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Maternal oxytocin treatment at birth increases epigenetic age in male offspring.

Exogenous oxytocin (OT) is widely used to induce or augment labor with little understanding of the impact on offspring development. In rodent models, including the prairie vole (Microtus ochrogaster), it has been shown that oxytocin administered to mothers can affect the nervous system of the offspring with long lasting behavioral effects especially on sociality. Here, we examined the hypothesis that perinatal oxytocin exposure could have epigenetic and transcriptomic consequences. Prairie voles were exposed to exogenous oxytocin, through injections given to the mother just prior to birth, and were studied at the time of weaning. The outcome of this study revealed increased epigenetic age in oxytocin-exposed animals compared to the saline-exposed group. Oxytocin exposure led to 900 differentially methylated CpG sites (annotated to 589 genes), and 2 CpG sites (2 genes) remained significantly different after correction for multiple comparisons. Differentially methylated CpG sites were enriched in genes known to be involved in regulation of gene expression and neurodevelopment. Using RNA-sequencing we also found 217 nominally differentially expressed genes (p<0.05) in nucleus accumbens, a brain region involved in reward circuitry and social behavior; after corrections for multiple comparisons 6 genes remained significantly differentially expressed. Finally, we found that maternal oxytocin administration led to widespread alternative splicing in the nucleus accumbens. These results indicate that oxytocin exposure during birth may have long lasting epigenetic consequences. A need for further investigation of how oxytocin administration impacts development and behavior throughout the lifespan is supported by these outcomes.

Expression of bond-related behaviors affects titi monkey responsiveness to oxytocin and vasopressin treatments.

Social bonds influence physiology and behavior, which can shape how individuals respond to physical and affective challenges. Coppery titi monkey (Plecturocebus cupreus) offspring form selective bonds with their fathers, making them ideal for investigating how father-daughter bonds influence juveniles' responses to oxytocin (OT) and arginine-vasopressin (AVP) manipulations. We quantified the expression of father-daughter bond-related behaviors in females (n = 10) and gave acute intranasal treatments of saline, low/medium/high OT, low/high AVP, or an OT receptor antagonist (OTA) to subjects prior to a parent preference test. While females spent more time in proximity to their parents than strangers, we found a large degree of individual variation. Females with greater expression of bonding behaviors responded to OT treatments in a dose-dependent manner. Subjects also spent less time in proximity to strangers when treated with High OT (p = 0.003) and Low OT (p = 0.007), but more time when treated with High AVP (p = 0.007), Low AVP (p = 0.009), and OTA (p = 0.001). Findings from the present study suggest that variation in the expression of bond-related behaviors may alter responsiveness to OT and AVP, increasing engagement with unfamiliar social others. This enhanced sociality with strangers may promote the formation of pair bonds with partners.

Hypothalamic Paraventricular Stimulation Inhibits Nociceptive Wide Dynamic Range Trigeminocervical Complex Cells via Oxytocinergic Transmission.

Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.

A dog's life: Early life histories influence methylation of glucocorticoid (NR3C1) and oxytocin (OXTR) receptor genes, cortisol levels, and attachment styles.

Early life deprivation and stress can contribute to life-long, problematic consequences, including epigenetic variations related to behavior and health. Domestic dogs share human environments and social-cognitive traits, making them a promising comparative model to examine developmental plasticity. We examined 47 owner-dog dyads, including dogs rescued from abusive or neglectful environments, and matched control dogs for changes in DNA methylation of glucocorticoid (NR3C1) and oxytocin (OXTR) receptor genes previously shown to be affected by early life stress in other species including humans. We used an attachment paradigm, which included a separation event to examine cortisol levels and owner-dog attachment styles. Overall, dogs with adverse histories had different NR3C1 methylation patterns as a function of age and less OXTR methylation than comparison dogs. Dogs with adverse histories did not differ in their cortisol change from baseline to poststressor from comparison dogs, but the change in cortisol was associated with NR3C1 methylation. In addition, dogs with a history of early life stress had more insecure attachment styles; for every unit increase of OXTR methylation, the odds increased for insecure attachment style. This study demonstrates that adverse life histories lead to methylation differences, resulting in the hypothalamic-pituitary-adrenal (HPA) axis's dysregulation and differences in behavioral phenotypes.

Postpartum Oxytocin Treatment via the Mother Reprograms Long-Term Behavioral Disorders Induced by Early Life Stress on the Plasma and Brain Metabolome in the Rat.

The rat model of perinatal stress (PRS), in which exposure of pregnant dams to restraint stress reduces maternal behavior, is characterized by a metabolic profile that is reminiscent of the "metabolic syndrome". We aimed to identify plasma metabolomic signatures linked to long-term programming induced by PRS in aged male rats. This study was conducted in the plasma and frontal cortex. We also investigated the reversal effect of postpartum carbetocin (Cbt) on these signatures, along with its impact on deficits in cognitive, social, and exploratory behavior. We found that PRS induced long-lasting changes in biomarkers of secondary bile acid metabolism in the plasma and glutathione metabolism in the frontal cortex. Cbt treatment demonstrated disease-dependent effects by reversing the metabolite alterations. The metabolomic signatures of PRS were associated with long-term cognitive and emotional alterations alongside endocrinological disturbances. Our findings represent the first evidence of how early life stress may alter the metabolomic profile in aged individuals, thereby increasing vulnerability to CNS disorders. This raises the intriguing prospect that the pharmacological activation of oxytocin receptors soon after delivery through the mother may rectify these alterations.

Effects of physical training on hypothalamic neuronal activation and expressions of vasopressin and oxytocin in SHR after running until fatigue.

To assess the influence of physical training on neuronal activation and hypothalamic expression of vasopressin and oxytocin in spontaneously hypertensive rats (SHR), untrained and trained normotensive rats and SHR were submitted to running until fatigue while internal body and tail temperatures were recorded. Hypothalamic c-Fos expression was evaluated in thermoregulatory centers such as the median preoptic nucleus (MnPO), medial preoptic nucleus (mPOA), paraventricular nucleus of the hypothalamus (PVN), and supraoptic nucleus (SON). The PVN and the SON were also investigated for vasopressin and oxytocin expressions. Although exercise training improved the workload performed by the animals, it was reduced in SHR and followed by increased internal body temperature due to tail vasodilation deficit. Physical training enhanced c-Fos expression in the MnPO, mPOA, and PVN of both strains, and these responses were attenuated in SHR. Vasopressin immunoreactivity in the PVN was also increased by physical training to a lesser extent in SHR. The already-reduced oxytocin expression in the PVN of SHR was increased in response to physical training. Within the SON, neuronal activation and the expressions of vasopressin and oxytocin were reduced by hypertension and unaffected by physical training. The data indicate that physical training counterbalances in part the negative effect of hypertension on hypothalamic neuronal activation elicited by exercise, as well as on the expression of vasopressin and oxytocin. These hypertension features seem to negatively influence the workload performed by SHR due to the hyperthermia derived from the inability of physical training to improve heat dissipation through skin vasodilation.

Effects of commonly used carbamates (carbaryl and thiram) on the regulatory, secretory and motor functions of bovine cervixes in vitro.

Previously studied classes of pesticides, including organochlorines, organophosphates and pyrethroids disturb the mechanism that causes bovine myometrial contractions. Hence, the aim of this study was to investigate the effects of carbaryl and thiram, which are representative carbamate pesticides commonly used in global agriculture, on the motor and secretory functions of bovine cervixes. Additionally, the impacts of these pesticides on intra- and intercellular signaling in vitro were estimated. In this study, cervical cells or strips were obtained from cows at days 18-20 of the estrous cycle and were treated with carbaryl or thiram. Neither carbamate (10 or 100 ng/ml) exerted cytotoxic effects. Carbaryl increased the level of mRNA (at a dose of 0.1 ng/ml) and protein (at both doses, 1 and 10 ng/ml) expression for the oxytocin receptor (OXTR), while thiram (at 0.1 and 10 ng/ml or 0.1-10 ng/ml, respectively) caused the opposite effects. Moreover, the level of the second messenger inositol-trisphosphate (IP3) was decreased by carbaryl (10 ng/ml) but increased by thiram (10 ng/ml). Only thiram decreased prostaglandin-endoperoxide synthase 2 (PTGS2; 0.1 ng/ml) and aldo-keto reductase family 1, member B1 (AKR1B1; 0.1 ng/ml), and prostaglandin E synthase 2 (PTGES2; 0.1-10 ng/ml) mRNA expression, while thiram (0.1-10 ng/ml) and carbaryl (0.1 and 10 ng/ml) both decreased the release of PGF2α. Carbaryl (10 ng/ml) and thiram (10 ng/ml) also decreased the level of a gap junction protein (GAP). Moreover, carbaryl (10 ng/ml) decreased the level of myosin light chain kinase (MLCK). However, the strength of cervical contractions was increased by thiram (1 and 10 ng/ml) but decreased by carbaryl (1 and 10 ng/ml). Carbaryl increased the receptivity of cervical cells to oxytocin (OXT), but inhibited further transduction (IP3) of this signal. Hence, direct inhibition of cervical strip contraction may occur. In contrast, thiram mostly decreased the receptivity of cervical cells to OXT, while it stimulated the contraction of cervical strips. Moreover, compared to carbaryl, thiram more greatly affected the synthesis and release of prostaglandins. These results suggest that carbaryl and thiram disturb OXT signaling, PG secretion and cervical contraction in vitro.

Cinnamic acid promotes elongation of hair peg-like sprouting in hair follicle organoids via oxytocin receptor activation.

Considerable global demand exists for the development of novel drugs for the treatment of alopecia. A recent report demonstrated that oxytocin promotes hair growth activity in human dermal papilla (DP) cells; however, its application in drugs or cosmetic products is challenging because rapid degradation and relatively large molecular weight prevent long-term topical administration on the scalp. Here, we examined cinnamic acid, a small molecule activator for oxytocin receptor (OXTR) expression. Treatment with cinnamic acid led to upregulation of OXTR and trichogenic gene expression in human DP cells. Furthermore, inhibition of OXTR with an antagonist, L-371,257, suppressed hair growth-related gene expression in DP cells. These findings suggest that cinnamic acid enhances the hair growth ability of DP cells via oxytocin signaling. Additionally, we tested the hair growth-promoting effects of cinnamic acid using hair follicle organoids in vitro and observed that cinnamic acid significantly promoted the growth of hair peg-like sprouting. These promising results may be useful for developing hair growth-promoting products targeting oxytocin.

Adolescent social isolation disrupts developmental tuning of neuropeptide circuits in the hypothalamus to amygdala regulating social and defensive behavior.

Engaging in positive social (i.e., prosocial) interactions during adolescence acts to modulate neural circuits that determine adult adaptive behavior. While accumulating evidence indicates that a strong craving for prosocial behavior contributes to sustaining neural development, the consequences of social deprivation during adolescence on social neural circuits, including those involving oxytocin (OXT) and vasopressin (AVP), are poorly characterized. We evaluated adaptive behaviors in socially isolated mice, including anxiety-like, social, and defensive behaviors, along with OXT and AVP neural profiles in relevant brain regions. Social isolation from postnatal day (P-)22 to P-48 induced enhanced defensive and exploratory behaviors, in nonsocial and social contexts. Unlike OXT neurons, AVP+ cell density in the paraventricular nucleus of the hypothalamus increases with age in males. Social isolation also modulated gene expression in the medial amygdala (MeA), including the upregulation of OXT receptors in males and the downregulation of AVP1a receptors in both sexes. Socially isolated mice showed an enhanced defensive, anogenital approach toward a novel adult female during direct social interactions. Subsequent c-Fos mapping revealed diminished neural activity in restricted brain areas, including the MeA, lateral septum, and posterior intralaminar nucleus of the thalamus, in socially isolated mice. These data indicate that neural signals arising from daily social interactions invoke region-specific modification of neuropeptide expression that coordinates with altered defensiveness and neural responsivities, including OXT- and AVP-projecting regions. The present findings indicate an involvement of OXT and AVP circuits in adolescent neural and behavioral plasticity that is tuned by daily social interaction.

Embryo-endometrial interaction associated with the location of the embryo during the mobility phase in mares.

Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.

Oxytocin alleviates periodontitis in adult rats.

OBJECTIVE: The objective of the study was to evaluate the expression of oxytocin receptors in normal and inflamed gingiva, as well as the effects of systemic administration of oxytocin in bone loss and gum inflammatory mediators in a rat model of experimental periodontitis. BACKGROUND DATA: Current evidence supports the hypothesis of a disbalance between the oral microbiota and the host's immune response in the pathogenesis of periodontitis. Increased complexity of the microbial biofilm present in the periodontal pocket leads to local production of nitrogen and oxygen-reactive species, cytokines, chemokines, and other proinflammatory mediators which contribute to periodontal tissue destruction and bone loss. Oxytocin has been suggested to participate in the modulation of immune and inflammatory processes. We have previously shown that oxytocin, nitric oxide, and endocannabinoid system interact providing a mechanism of regulation for systemic inflammation. Here, we aimed at investigating not only the presence and levels of expression of oxytocin receptors on healthy and inflamed gingiva, but also the effects of oxytocin treatment on alveolar bone loss, and systemic and gum expression of inflammatory mediators involved in periodontal tissue damage using ligature-induced periodontitis. Therefore, anti-inflammatory strategies oriented at modulating the host's immune response could be valuable adjuvants to the main treatment of periodontal disease. METHODS: We used an animal model of ligature-induced periodontitis involving the placement of a linen thread (Barbour flax 100% linen suture, No. 50; size 2/0) ligature around the neck of first lower molars of adult male rats. The ligature was left in place during the entire experiment (7 days) until euthanasia. Animals with periodontitis received daily treatment with oxytocin (OXT, 1000 µg/kg, sc.) or vehicle and/or atosiban (3 mg/kg, sc.), an antagonist of oxytocin receptors. The distance between the cement-enamel junction and the alveolar bone crest was measured in stained hemimandibles in the long axis of both buccal and lingual surfaces of both inferior first molars using a caliper. TNF-α levels in plasma were determined using specific rat enzyme-linked immunosorbent assays (ELISA). OXT receptors, IL-6, IL-1ß, and TNF-α expression were determined in gingival tissues by semiquantitative or real-time PCR. RESULTS: We show that oxytocin receptors are expressed in normal and inflamed gingival tissues in male rats. We also show that the systemic administration of oxytocin prevents the experimental periodontitis-induced increased gum expression of oxytocin receptors, TNF-α, IL-6, and IL-1ß (p < .05). Furthermore, we observed a reduction in bone loss in rats treated with oxytocin in our model. CONCLUSIONS: Our results demonstrate that oxytocin is a novel and potent modulator of the gingival inflammatory process together with bone loss preventing effects in an experimental model of ligature-induced periodontitis.

Methylation of the Oxytocin, Oxytocin Receptor, and Vasopressin Gene Promoters in Tobacco Use Disorder during Cessation.

INTRODUCTION: Vasopressin (AVP) and oxytocin (OT) exert sex-specific effects on social pair bonding and stress reactions while also influencing craving in substance use disorders. In this regard, intranasal oxytocin (OT) and AVP antagonists present potential treatments for tobacco use disorder (TUD). Since transcription of both hormones is also regulated by gene methylation, we hypothesized sex-specific changes in methylation levels of the AVP, OT, and OT receptor (OXTR) gene during nicotine withdrawal. METHODS: The study population consisted of 49 smokers (29 males, 20 females) and 51 healthy non-smokers (25 males, 26 females). Blood was drawn at day 1, day 7, and day 14 of smoking cessation. Craving was assessed with the questionnaire on smoking urges (QSU). RESULTS: Throughout cessation, mean methylation of the OT promoter gene increased in males and decreased in females. OXTR receptor methylation decreased in females, while in males it was significantly lower at day 7. Regarding the AVP promoter, mean methylation increased in males while there were no changes in females. Using mixed linear modeling, CpG position, time point, sex, and the interaction of time point and sex as well as time point, sex, and QSU had a significant fixed effect on OT and AVP gene methylation. The interaction effect suggests that sex, time point, and QSU are interrelated, meaning that, depending on the sex, methylation could be different at different time points and vice versa. There was no significant effect of QSU on mean OXTR methylation. DISCUSSION: We identified differences at specific CpGs between controls and smokers in OT and AVP and in overall methylation of the AVP gene. Furthermore, we found sex-specific changes in mean methylation levels of the mentioned genes throughout smoking cessation, underlining the relevance of sex in the OT and vasopressin system. This is the first study on epigenetic regulation of the OT promoter in TUD. Our results have implications for research on the utility of the AVP and OT system for treating substance craving. Future studies on both targets need to analyze their effect in the context of sex, social factors, and gene regulation.

Male rat hypothalamic extraretinal photoreceptor Opsin3 is sensitive to osmotic stimuli and light.

The light-sensitive protein Opsin 3 (Opn3) is present throughout the mammalian brain; however, the role of Opn3 in this organ remains unknown. Since Opn3 encoded mRNA is modulated in the supraoptic and paraventricular nucleus of the hypothalamus in response to osmotic stimuli, we have explored by in situ hybridization the expression of Opn3 in these nuclei. We have demonstrated that Opn3 is present in the male rat magnocellular neurones expressing either the arginine vasopressin or oxytocin neuropeptides and that Opn3 increases in both neuronal types in response to osmotic stimuli, suggesting that Opn3 functions in both cell types and that it might be involved in regulating water balance. Using rat hypothalamic organotypic cultures, we have demonstrated that the hypothalamus is sensitive to light and that the observed light sensitivity is mediated, at least in part, by Opn3. The data suggests that hypothalamic Opn3 can mediate a light-sensitive role to regulate circadian homeostatic processes.

Neurophysin I is an analytically robust surrogate biomarker for oxytocin.

Oxytocin is a pleiotropic neuropeptide that plays roles in biological processes ranging from birth, lactation, and social bonding to immune function, cardiovascular repair, and regulation of appetite. Although measurements of endogenous oxytocin concentrations have been performed for more than 50 years, the ability to measure oxytocin accurately poses notable challenges. One potential solution for overcoming these challenges involves measurement of oxytocin's carrier molecule - neurophysin I (NP-1) - as a surrogate biomarker. NP-1 is secreted in equimolar concentrations with oxytocin but has a longer half-life, circulates in higher concentrations, and can be measured using a sandwich immunoassay. We report experiments that 1) analytically validate a commercially available NP-1 sandwich immunoassay for use with human plasma and urine samples, 2) confirm the specificity of this assay, based on detection of NP-1 in plasma from wild-type but not oxytocin knockout mice, 3) demonstrate that NP-1 concentrations are markedly elevated in late pregnancy, consistent with studies showing substantial increases in plasma oxytocin throughout gestation, and 4) establish strong correlation between NP-1 and plasma oxytocin concentrations when oxytocin is measured in extracted (but not non-extracted) plasma. The NP-1 assay used in this study has strong analytical properties, does not require time-intensive extraction protocols, and the assay itself can be completed in < 2 h (compared to 16-24 h for a competitive oxytocin immunoassay). Our findings suggest that much like copeptin has become a useful surrogate biomarker in studies of vasopressin, measurements of NP-1 have similar potential to advance oxytocin research.