Oxytocin Receptor Expression Research Articles

Oxytocin excites BNST interneurons and inhibits BNST output neurons to the central amygdala

The dorsolateral bed nucleus of the stria terminalis (BNSTDL) has high expression of oxytocin (OT) receptors (OTR), which were shown to facilitate cued fear. However, the role of OTR in the modulation of BNSTDL activity remains elusive. BNSTDL contains GABA-ergic neurons classified based on intrinsic membrane properties into three types. Using in vitro patch-clamp recordings in male rats, we demonstrate that OT selectively excites and increases spontaneous firing rate of Type I BNSTDL neurons. As a consequence, OT increases the frequency, but not amplitude, of spontaneous inhibitory post-synaptic currents (sIPSCs) selectively in Type II neurons, an effect abolished by OTR antagonist or tetrodotoxin, and reduces spontaneous firing rate in these neurons. These results suggest an indirect effect of OT in Type II neurons, which is mediated via OT-induced increase in firing of Type I interneurons. As Type II BNSTDL neurons were shown projecting to the central amygdala (CeA), we also recorded from retrogradely labeled BNST→CeA neurons and we show that OT increases the frequency of sIPSC in these Type II BNST→CeA output neurons. In contrast, in Type III neurons, OT reduces the amplitude, but not frequency, of both sIPSCs and evoked IPSCs via a postsynaptic mechanism without changing their intrinsic excitability. We present a model of fine-tuned modulation of BNSTDL activity by OT, which selectively excites BNSTDL interneurons and inhibits Type II BNST→CeA output neurons. These results suggest that OTR in the BNST might facilitate cued fear by inhibiting the BNST→CeA neurons.

The Endometrium Contributes to the Differential Contractile Response to Oxytocin of Uterine Tissue from Virgin and Proven Breeder Non‐pregnant Rats

Introduction We hypothesized that the hormonal and physical stimuli experienced by the uterus during pregnancy induce epigenetic modifications that affect the physiological performance and health status of this organ. We have previously shown that the contractile response of the uterus from virgin (V) and proven breeder (PB) rats to oxytocin is different. Here we investigated the impact of the endometrium in this response. Methods Virgin (V) and proven breeder (PB) NP female 18 week-old rats were staged and euthanized at proestrus (IACUC #2758). Uterine horns were dissected in strips, suspended in a tissue bath containing Krebs solution at 37°C, and bubbled with 95% O2 and 5% CO2. Contractility of tissue in response to increasing levels of oxytocin (10-10 to 10-5 M) were recorded. Oxytocin receptor (OXTR) mRNA expression was evaluated withTaqman® gene expression assays (Life Technologies) and QuantStudio™ 5 real-time PCR system (Applied Biosystems, Grand Island, NY). Distribution of OXTR in the myometrium and endometrium was detected via immunohistochemistry(Primaries: OXTR antibody (BIOSS #bs-1314R) and primary β-actin antibody (Santa Cruz Biotechnology INC #sc-47778). Secondaries: Sheep anti-rabbit FITC, Sigma Aldrich #F7512 and donkey anti-mouse, Alexa Fluor 594 –(Invitrogen, #A21203) Dilution 1:200). Samples were mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories #H1800) and observed with a Nikon A1R Laser Scanning Confocal Microscope. Results Immunohistochemical imaging of the uterine samples we used in our motility experiments revealed a significant presence of endometrium. Thus, we hypothesized that previously observed differences in the response of V and PB tissue to oxytocin might be in part caused by the endometrium. Dose response to oxytocin performed on tissue fully stripped of the endometrium showed that V samples had stronger responses to oxytocin than PB samples (P<0.01). In particular, PB samples showed a significantly lower range of response (max-min) than their unstripped counterparts. In V samples, both maximal response and response range were more elevated than in unstripped counterparts (P<0.01). qRT-PCR did not showed any significant difference in the expression of OXTR in the endometrium of V and PB. Hence, the differences in the endometrial control of uterine contractility observed in the two groups are likely determined by other factors. Conclusions This study showed that the complete removal of the endometrium significantly alters the differences previously observed in the contractile response to oxytocin. Although no parity-related difference in the expression of the OXTR was detected in the endometrium, we speculate that other aspects of the endometrial regulation of uterine smooth muscles might be altered by pregnancy.

Effects of Oxytocin Analogs on Neurite Outgrowth and Cellular Signaling in Human SH‐SY5Y Neuroblastoma Cell Line

Introduction In the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G-protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression. Methods The human neuroblastoma derived SH-SY5Y cell line demonstrates morphological characteristics of neurons including neurites, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8-AM to measure ligand-induced Ca2+mobilization. Sigmoidal dose-response curves for OT analogs were generated using three-parameter nonlinear regression analysis in GraphPad Prism 8.3 software. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of the consensus mammalian OT sequence (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly; Leu8-OT) and a new world monkey analog (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Pro-Gly; Pro8-OT) for 24 hours, fixed and stained with phalloidin and DAPI. The Nikon A1R confocal microscope was used to collect 60X images of GFP-expressing neurons assessed for effects on neurite outgrowth using the Image J package FIJI with the NeuronJ plugin. Results In SH-SY5Y cells, both OT analogs were more potent than AVP at inducing Ca2+ mobilization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L-371,257. Additionally, both OT analogs show a dose-dependent increase in neurite outgrowth. Conclusions Together, these data suggest that OTR mediated signaling pathways lead to neurite outgrowth in SH-SY5Y cells. Integrative studies of behavior, genetics and ligand-receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.

Oxytocin Receptors Inhibit Output Neurons of the Bed Nucleus of the Stria Terminalis (BNST) and Promote Fear Responses to Predictable Threats

The dorsolateral bed nucleus of the stria terminalis (BNSTDL) has high expression of oxytocin (OT) receptors (OTR), which were shown to facilitate cued fear measured in the fear-potentiated startle. However, the mechanisms of these OT effects in the BNSTDL remain elusive.

Developing Brain Glucose Transporters, Serotonin, Serotonin Transporter, and Oxytocin Receptor Expression in Response to Early-Life Hypocaloric and Hypercaloric Dietary, and Air Pollutant Exposures

Perturbed maternal diet and prenatal exposure to air pollution (AP) affect the fetal brain, predisposing to postnatal neurobehavioral disorders. Glucose transporters (GLUTs) are key in fueling neurotransmission; deficiency of the neuronal isoform GLUT3 culminates in autism spectrum disorders. Along with the different neurotransmitters, serotonin (5-HT) and oxytocin (OXT) are critical for the development of neural connectivity. Serotonin transporter (SERT) modulates synaptic 5-HT levels, while the OXT receptor (OXTR) mediates OXT action. We hypothesized that perturbed brain GLUT1/GLUT3 regulated 5-HT-SERT imbalance, which serves as a contributing factor to postnatal neuropsychiatric phenotypes, with OXT/OXTR providing a counterbalance. Employing maternal diet restriction (intrauterine growth restriction [IUGR]), high-fat (HF) dietary modifications, and prenatal exposure to simulated AP, fetal (E19) murine brain 5-HT was assessed by ELISA with SERT and OXTR being localized by immunohistochemistry and measured by quantitative Western blot analysis. IUGR with lower head weights led to a 48% reduction in male and female fetal brain GLUT3 with no change in GLUT1, when compared to age- and sex-matched controls, with no significant change in OXTR. In addition, a ∼50% (p = 0.005) decrease in 5-HT and SERT concentrations was displayed in fetal IUGR brains. In contrast, despite emergence of microcephaly, exposure to a maternal HF diet or AP caused no significant changes. We conclude that in the IUGR during fetal brain development, reduced GLUT3 is associated with an imbalanced 5-HT-SERT axis. We speculate that these early changes may set the stage for altering the 5HT-SERT neural axis with postnatal emergence of associated neurodevelopmental disorders.

Decidual cell FKBP51–progesterone receptor binding mediates maternal stress–induced preterm birth

Depression and posttraumatic stress disorder increase the risk of idiopathic preterm birth (iPTB); however, the exact molecular mechanism is unknown. Depression and stress-related disorders are linked to increased FK506-binding protein 51 (FKBP51) expression levels in the brain and/or FKBP5 gene polymorphisms. Fkbp5-deficient (Fkbp5-/-) mice resist stress-induced depressive and anxiety-like behaviors. FKBP51 binding to progesterone (P4) receptors (PRs) inhibits PR function. Moreover, reduced PR activity and/or expression stimulates human labor. We report enhanced in situ FKBP51 expression and increased nuclear FKBP51-PR binding in decidual cells of women with iPTB versus gestational age-matched controls. In Fkbp5+/+ mice, maternal restraint stress did not accelerate systemic P4 withdrawal but increased Fkbp5, decreased PR, and elevated AKR1C18 expression in uteri at E17.25 followed by reduced P4 levels and increased oxytocin receptor (Oxtr) expression at 18.25 in uteri resulting in PTB. These changes correlate with inhibition of uterine PR function by maternal stress-induced FKBP51. In contrast, Fkbp5-/- mice exhibit prolonged gestation and are completely resistant to maternal stress-induced PTB and labor-inducing uterine changes detected in stressed Fkbp5+/+ mice. Collectively, these results uncover a functional P4 withdrawal mechanism mediated by maternal stress-induced enhanced uterine FKBP51 expression and FKPB51-PR binding, resulting in iPTB.

Involvement of Protein Kinase A in Oxytocin Neuronal Activity in Rat Dams with Pup Deprivation.

Oxytocin (OT) neuronal activity is the key factor for breastfeeding and it can be disrupted by mother-baby separation. To explore cellular mechanisms underlying OT neuronal activity, we studied the role of protein kinase A (PKA) in OT neuronal activity in the supraoptic nucleus (SON) using a rodent model of pup deprivation (PD) Intermittent (IPD) or continuous (CPD) PD significantly reduced suckling duration and number of milkejections in lactating rats, particularly those with CPD. In Western blots of the SON, PD increased expressions of OT receptor (OTR) and its immediate downstream effectors, Gαq and Gβ subunits, particularly IPD, but reduced the expression of catalytic subunit of PKA (cPKA). In brain slices, inhibition of PKA blocked prostaglandin E2-evoked increase in firing activity including burst firing in OT neurons. In IPD dams, filamentous actin formed ring-like structures in the cytoplasmic region of OT neurons, which was reduced in CPD. Moreover, molecular association between actin and cPKA also reduced in PD dams. Incubation of brain slices with OT reduced the expression of cPKA, which was blocked by pretreatment with atosiban, an antagonist of OTR. These results indicate that PD disrupts OT neuronal activity through dissociating the Gq proteins and PKA in OTR-associated signaling cascade, which couples with reduced interactions between filamentous actin and PKA in OT neurons in the SON. This study highlights that PKA can be a novel target treating abnormal OT neuronal activity and its associated diseases.

ΔMST and the Regulation of Cardiac CSE and OTR Expression in Trauma and Hemorrhage.

Genetic deletion of 3-mercaptopyruvate sulfurtransferase (MST) is known to result in hypertension and cardiac hypertrophy in older mice, and is associated with increased anxiety-like behaviors. Endogenous hydrogen sulfide (H2S) produced by MST in the mitochondria is also known to be involved in physiological and cellular bioenergetics, and its dysfunction associated with depressive behavior and increased cardiovascular morbidity. Interestingly, early life stress has been shown to lead to a significant loss of cystathionine-γ-lyase (CSE) and oxytocin receptor (OTR) expression in the heart. Thus, we were interested in testing the hypothesis of whether genetic MST mutation (ΔMST) would affect cardiac CSE and OTR expression and affect the mitochondrial respiration in a clinically relevant, resuscitated, mouse model of trauma and hemorrhagic shock. In ΔMST mice, we found a reduction of CSE and OTR in both the naive as well as injured state, in contrast to the wild type (wt) controls. Interestingly, the ΔMST showed a different complex IV response to injury than the wt controls, although our claims are based on the non-demonstrated assumption that naive wt and naive ΔMST mice have comparable complex IV activity. Finally, hemorrhagic shock led to a reduction of CSE and OTR, confirming previous results in the injured mouse heart. To date, the exact mechanisms of the cardiac interaction between H2S and OT are not clear, but they point the way to potential cardioprotective therapies.

Intracranial mast cells contribute to the control of social behavior in male mice

Mast cells (MCs) exist intracranially and have been reported to affect higher brain functions in rodents. However, the role of MCs in the regulation of emotionality and social behavior is unclear. In the present study, using male mice, we examined the relationship between MCs and social behavior and investigated the underlying mechanisms. Wild-type male mice intraventricularly injected with a degranulator of MCs exhibited a marked increase in a three-chamber sociability test. In addition, removal of MCs in Mast cell-specific Toxin Receptor-mediated Conditional cell Knock out (Mas-TRECK) male mice showed reduced social preference levels in a three-chamber sociability test without other behavioral changes, such as anxiety-like and depression-like behavior. Mas-TRECK male mice also had reduced serotonin content and serotonin receptor expression and increased oxytocin receptor expression in the brain. These results suggested that MCs may contribute to the regulation of social behavior in male mice. This effect may be partially mediated by serotonin derived from MCs in the brain.

Oxytocin receptor expression in the midbrain dorsal raphe is dynamic across female reproduction in rats.

Central oxytocin receptor (OTR) expression is extremely sensitive to circulating steroid hormones and OTRs influence many of the neurobehavioural adaptations associated with female reproduction (e.g., postpartum caregiving, aggression, cognition, affective responses). Changes in central OTR expression across female reproduction have often been studied, but almost all of such research has focused on the forebrain, ignoring hormone-sensitive midbrain sites such as the serotonergic dorsal raphe (DR) that are also critical for postpartum behaviours. To investigate the effects of female reproductive state on OTRs in the DR, we first used autoradiography to examine OTR binding across four female reproductive states in laboratory rats: dioestrous virgin, pregnancy day 10, the day of parturition and postpartum day 7. OTR binding in the rostral DR (but not other DR subregions) was approximately 250% higher in parturient rats compared to dioestrous virgins and dropped back down to virgin levels by postpartum day 7. Given the chemical heterogeneity of the DR, we then examined OTR expression in the three most abundant neuronal phenotypes of the DR (i.e., serotonin, GABA and dopamine) in dioestrous virgins and recently parturient females. Using dual-label immunohistochemistry and in situ hybridisation, we found that twice as many dopaminergic cells in the parturient rostral DR contained OTR immunoreactivity compared to that found in virgins. On the other hand, mothers had fewer rostral DR GABAergic cells expressing OTRs than did virgins. OTR expression in serotonin cells did not differ between the two groups. Overall, these results suggest that the rostral subregion of the midbrain DR is uniquely sensitive to oxytocin around the time of parturition, with subpopulations of cells that become more sensitive (i.e., dopamine), less sensitive (i.e., GABA) and show no change (i.e., serotonin) to this neuropeptide. This dynamic OTR signalling in the female DR may help drive the numerous behavioural changes across female reproduction that are necessary for successful motherhood.

Oxytocin receptor expression is associated with estrogen receptor status in breast tumors

The oxytocin receptor (OXTR) plays an important role in childbirth, breastfeeding, and social interactions. There is emerging evidence that OXTR is associated with the breast cancer (BC) initiation and progression. However, the mechanisms leading to a change in its expression, the diagnostic or prognostic value of the receptor in BC are currently poorly understood. Here, we have evaluated the relative level of OXTR expression in BC samples (n=107), and also investigated the effect of estradiol on its expression in MCF-7 and MDA-MB-231 cells. The level of OXTR expression was significantly lower in breast tumor tissue than in normal tissue obtained from the same patient. The expression of OXTR was dependent on the status and expression of the estrogen receptor (ER): the level of OXTR mRNA was significantly lower in ER-negative BC samples compared to ER-positive BC samples. Moreover, OXTR expression was also lower in samples from patients with luminal subtype with a low value of ER expression (0-5 score according to the IHC assay, Allred scoring) compared with samples with high ER expression (6-8 score). In luminal BC, OXTR expression was associated with the HER2 expression level: the OXTR mRNA level was higher in tumors with a HER2 IHC score of 1+ as compared to cases with the HER2 expression score of 2+, 3+. We also showed that estradiol increased the level of OXTR mRNA in MCF-7 cells, but not in ER-negative MDA-MB-231 cells. These data indicate that changes in OXTR expression in BC tissues can be caused by increased ER expression. We found no association between OXTR and T or N stages and progesterone receptor expression.

Uterine receptor activation in response to uterotonic agents in late-pregnancy rat myometrium following pretreatment with oxytocin: an in vitro study

Background: Long term exposure to oxytocin reduces the ability of myometrium to respond to oxytocin, leading to oxytocin receptor (OXTR) desensitization. In this study we analyzed the response to other uterotonics such as prostaglandin, as well as investigating prostaglandin E2 receptors (EP3) and prostaglandin F2α receptors (FP). We hypothesized that compensatory mechanisms would increase the expression and activation of FP and EP3 following OXTR desensitization. Methods: Myometrium from late-pregnancy rats was collected in order to assess mRNA expression levels for OXTR, FP, and EP3 using RT-PCR. This was done after 2 hours of pretreatment with 10-6 M oxytocin to induce OXTR desensitization, or equilibration in physiological salt solution (PSS). Myometrium was exposed to increasing concentrations of uterotonic agents (10-10 to 10-5 M) following 2 hours of pretreatment with 10-6 M oxytocin (experimental group) or with PSS (control group). Myometrium from the experimental group was washed with PSS and OXTR expression was assessed using Western blot and RT-PCR. Results: mRNA expression levels for EP3, FP and OXTR were not statistically different between the experimental (OXTR desensitization) and control groups. Compared to the control group, the (mean ± SD) contractile potency of carboprost (pEC50: 7.74 ± 0.56 vs 6.81 ± 0.25, P = 0.03) and maximal contractility of misoprostol (Emax(ratio): 4.44 ± 3.60 vs 1.32 ± 0.22, P = 0.02) were significantly increased in the OXTR desensitization group, while the contractility of oxytocin was significantly reduced (Emax(ratio): 1.62 ± 0.27 vs 2.82 ± 0.98, P = 0.015). No significant differences in myometrial OXTR expression were observed between the PSS, carboprost and misoprostol groups following OXTR desensitization. Discussion: Following OXTR desensitization of myometrium, FP and EP3 activation increased in a compensatory manner, but not FP and EP3 receptor expression.

Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

Commensal microbe-derived propionic acid mediates juvenile social isolation-induced social deficits and anxiety-like behaviors

Social experiences during early life are thought to be critical for proper social and emotional development. Conversely, social insults during development causes long-lasting behavioral abnormalities later in life. However, how juvenile social deprivation influences social and emotional behaviors remains poorly understood. Here, we show that juvenile social isolation induces a shift in microbial ecology that negatively impacts social and emotional behaviors in adulthood. These behavioral changes, which occur during this critical period are transferable to antibiotic pre-treated mice by fecal microbiota transplant. In addition, juvenile social isolation decreases the expression of oxytocin receptor (OXTR) in the medial prefrontal cortex (mPFC), and increases the amounts of fecal propionic acid (PA), a short-chain fatty acid derived from gut micobiota. Accordingly, infusion with an OXTR antagonist (OXTR-A, l-368,899) specifically in the mPFC or supplementation of PA both can cause social deficits and anxiety-like behaviors in group housed mice. Collectively, our findings reveal that juvenile social experience regulates prefrontal cortical OXTR expression through gut microbiota-produced PA and that is essential for normal social and emotional behaviors, thus providing a cellular and molecular context to understand the consequences of juvenile social deprivation.

The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression.

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined.Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour.These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

Increased Response to 3,4-Methylenedioxymethamphetamine (MDMA) Reward and Altered Gene Expression in Zebrafish During Short- and Long-Term Nicotine Withdrawal.

An interactive effect between nicotine and 3,4-methylenedioxymethamphetamine (MDMA) has been reported but the mechanism underlying such interaction is not completely understood. This study used zebrafish to explore gene expression changes associated with altered sensitivity to the rewarding effects of MDMA following 2-week exposure to nicotine and 2-60 days of nicotine withdrawal. Reward responses to MDMA were assessed using a conditioned place preference (CPP) paradigm and gene expression was evaluated using quantitative real-time PCR of mRNA from whole brain samples from drug-treated and control adult zebrafish. Zebrafish pre-exposed for 2 weeks to nicotine showed increased conditioned place preference in response to low-dose, 0.1 mg/kg, MDMA compared to un-exposed fish at 2, 7, 30 and 60 days withdrawal. Pre-exposure to nicotine for 2 weeks induced a significant increase of c-Fos and vasopressin receptor expression but a decrease of D3 dopaminergic and oxytocin receptor expression at 2 days of withdrawal. C-Fos mRNA increased also at 7, 30, 60 days of withdrawal. Nicotine pre-exposed zebrafish submitted to MDMA-induced CPP showed an increase in expression of p35 at day 2, α4 at day 30, vasopressin at day 7 and D3 dopaminergic receptor at day 7, 30 and 60. These gene alterations could account for the altered sensitivity to the rewarding effects of MDMA in nicotine pre-exposed fish, suggesting that zebrafish have an altered ability to modulate behaviour as a function of reward during nicotine withdrawal.

Aberrant expression of oxytocin receptor in endometrium and decidua in women who have experienced recurrent implantation failure

To detect the oxytocin receptor (OTR) expression levels in the endometrium and decidua from women who have experienced recurrent implantation failure (RIF) and fertile women.Laboratory study using human endometrial and decidual samples.University-affiliated hospital.Six patients with RIF and six fertile women were recruited for endometrial sampling on day 20-24 of the menstrual cycle. Decidual tissues were collected from women who had a history of RIF and experienced a spontaneous abortion at 6-8 weeks of gestation (n = 8) and women with healthy pregnancies that terminated for nonmedical reasons (n = 8).None.OTR expression in the endometrial and decidual tissues was detected with the use of real-time quantitative polymerase chain reaction and Western blotting.OTR protein and mRNA were significantly increased in the endometria of RIF patients. In the decidua, OTR protein was significantly up-regulated in the RIF group, whereas mRNA was significantly decreased in this group.Women who experienced RIF presented with an aberrant expression pattern of OTR in the endometria and decidua.

Involvement of Oxytocin Receptor/Erk/MAPK Signaling in the mPFC in Early Life Stress-Induced Autistic-Like Behaviors.

The neonatal or infant period is a critical stage for the development of brain neuroplasticity. Early life stresses in the neonatal period, including neonatal maternal separation (NMS), have adverse effects on an increased risk of psychiatric disorders in juveniles and adults. However, the underlying molecular mechanisms are not largely understood. Here, we found that juvenile rats subjected to 4 h daily NMS during postnatal days 1 to 20 exhibited autistic-like behavioral deficits without impairments in learning and memory functions. Molecular mechanism studies showed that oxytocin receptor (OXTR) in the medial prefrontal cortex of NMS rats was evidently downregulated when compared with control pups, especially in neurons. Erk/MAPK signaling, the downstream coupling signaling of OTXR, was also inhibited in NMS juvenile rats. Treatment with oxytocin could relieve NMS-induced social deficit behaviors and activated phosphorylation of Erk/MAPK signaling. Furthermore, medication with the inhibitor of H3K4 demethylase alleviated the abnormal behaviors in NMS rats and increased the expression of OXTR in the medial prefrontal cortex, which showed an epigenetic mechanism underlying social deficits induced by NMS. Taken together, these findings identified a molecular mechanism by which disruptions of mother–infant interactions influenced later displays of typical social behaviors and suggested the potential for NMS-driven epigenetic tuning of OXTR expression.

Gender-Dependent Effects of Thimerosal on Human Progenitor Neurons: A Potential Link to Regressive Autism

Regressive Autism [RA] is a subtype of autism spectrum disorder (ASD) that manifests as the loss of previously acquired developmental milestones and skills. Early life dysregulation of neurodevelopment due to exposure to toxic metals has been associated with ASD, but the underlying biological mechanisms by which metals influence neurodevelopment remain unclear. We explored the potential role of thimerosal or ethylmercury on neurite formation and oxytocin receptor (OXTR) modulation in four human-developing neuronal cell lines of male and female origin (N = 2 each). We exposed the cell lines to three levels of thimerosal that closely represented the concentrations an infant (equivalent to 1 L of blood volume), an adolescent (~5 L), and an adult (~10 L) might receive from multiple doses of vaccine containing 100 µg/mL of thimerosal. We found that exposure to vaccine-equivalent concentrations of thimerosal induced significantly greater neurite dysregulation, including central chromatolysis, neurite abnormalities (i.e., axonal length, relative diameter, and pathways in neurons), and syncytia formation in undifferentiated and partially differentiated human developing neurons compared to controls. Exposure of male neurons to thimerosal significantly affected neurite formation and OXTR expression compared to the female neurons. In developed nations, most vaccines are thimerosal-free; nonetheless, some still incorporate ethylmercury as a preservative. In contrast, due to the lack of refrigeration in many developing nations, thimerosal is still used widely in most vaccines. Our study shows that ethylmercury induced profound neurite dysregulation and downregulated OXTR expression. The progenitor neurons from males were significantly more susceptible to thimerosal than those from females. However, internal factors in vaccine recipients may trigger dysfunction in the Blood-Brain Barrier (BBB), and screening potentially vulnerable individuals for conditions that may contribute to a BBB breach before vaccination might be beneficial.