Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells.

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.

Antidepressant- and anxiolytic-like activities of Rosmarinus officinalis extract in rodent models: Involvement of oxytocinergic system.

BACKGROUND: Oxytocin (OXT), a neuropeptide involved in mammal reproductive and prosocial behaviors, has been reported to interact with various stressor-provoked neurobiological changes, including neuroendocrine, neurotransmitter, and inflammatory processes. In view of disturbances in psychosocial relationships due to social isolation and physical distancing measures amid the COVID-19 pandemic, being one of the triggering factors for the recent rise in depression and anxiety, OXT is a potential candidate for a new antidepressant. METHODS: In this present study, we have aimed to investigate the effects of oral administration of Rosmarinus officinalis extract (RE), extracted from distillation residue of rosemary essential oil, on central OXT level in the context of other stress biomarkers and neurotransmitter levels in mice models. Tail suspension test (TST) and elevated plus maze test (EPMT) following LPS injection were employed to assess depressive- and anxiety-like behavior in mice, respectively. FINDINGS: Pretreatment with RE for seven days significantly improved behavior in TST and EPMT. Whole-genome microarray analysis reveals that RE significantly reversed TST stress-induced alterations in gene expressions related to oxytocinergic and neurotransmitter pathways and inflammatory processes. In both models, RE significantly increased central Oxt and Oxtr expressions, as well as OXT protein levels. RE also significantly attenuated stress-induced changes in serum corticosterone, brain and serum BDNF levels, and brain neurotransmitters levels in both models. INTERPRETATION: Altogether, our study is the first to report antidepressant- and anxiolytic-like activities of RE through modulating oxytocinergic system in mice brain and thus highlights the prospects of RE in the treatment of depressive disorders of psychosocial nature.

Oxytocin in the Male Reproductive Tract; The Therapeutic Potential of Oxytocin-Agonists and-Antagonists.

In this review, the role of oxytocin and oxytocin-like agents (acting via the oxytocin receptor and belonging to the oxytocin-family) in the male reproductive tract is considered. Previous research (dating back over 60 years) is revised and connected with recently found aspects of the role oxytocin plays in male reproductive health. The local expression of oxytocin and its receptor in the male reproductive tract of different species is summarized. Colocalization and possible crosstalk to other agents and receptors and their resulting effects are discussed. The role of the newly reported oxytocin focused signaling pathways in the male reproductive tract, other than mediating contractility, is critically examined. The structure and effect of the most promising oxytocin-agonists and -antagonists are reviewed for their potential in treating male disorders with origins in the male reproductive tract such as prostate diseases and ejaculatory disorders.

Oxytocin in the anterior cingulate cortex is involved in helping behaviour.

Empathy toward the distress of others is thought to motivate helping behaviour, in the form of voluntary action to eliminate that distress. Neuropeptide oxytocin is associated with various social cognitive abilities, including empathy and prosocial behaviour. The anterior cingulate cortex is known to be one of the brain regions underlying empathy, and one in which oxytocin receptors are expressed. However, the relationship between helping behaviour and oxytocin in the anterior cingulate cortex is still unclear. The present study investigated whether oxytocin in the anterior cingulate cortex is involved in rats' helping behaviour. In Experiment 1, we examined the influence of blockading the oxytocin receptors in the anterior cingulate cortex on helping behaviour. Impeding oxytocin in the anterior cingulate cortex delayed learning of the helping behaviour. In Experiment 2, we examined immunofluorescent colocalization of oxytocin receptors and c-fos proteins in the anterior cingulate cortex, the anterior insular cortex, and the amygdala in rats that acquired helping behaviour. We found increased c-fos expression in oxytocin receptor-containing neurons in the anterior cingulate cortex and amygdala when the rats acquired helping behaviour. In addition, the change in neural activation was found in the late phase of the learning. These results suggest that the oxytocin in the cingulate-amygdala pathways may play an important role in helping behaviour.

I8-arachnotocin-an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor.

The neuropeptides oxytocin (OT) and vasopressin (VP) and their G protein-coupled receptors OTR, V1aR, V1bR, and V2R form an important and widely-distributed neuroendocrine signaling system. In mammals, this signaling system regulates water homeostasis, blood pressure, reproduction, as well as social behaviors such as pair bonding, trust and aggression. There exists high demand for ligands with differing pharmacological profiles to study the physiological and pathological functions of the individual receptor subtypes. Here, we present the pharmacological characterization of an arthropod (Metaseiulus occidentalis) OT/VP-like nonapeptide across the human OT/VP receptors. I8-arachnotocin is a full agonist with respect to second messenger signaling at human V2R (EC50 34 nM) and V1bR (EC50 1.2 µM), a partial agonist at OTR (EC50 790 nM), and a competitive antagonist at V1aR [pA2 6.25 (558 nM)]. Intriguingly, I8-arachnotocin activated the Gαs pathway of V2R without recruiting either ß-arrestin-1 or ß-arrestin-2. I8-arachnotocin might thus be a novel pharmacological tool to study the (patho)physiological relevance of ß-arrestin-1 or -2 recruitment to the V2R. These findings furthermore highlight arthropods as a novel, vast and untapped source for the discovery of novel pharmacological probes and potential drug leads targeting neurohormone receptors.

Oxytocin antagonism prevents pregnancy-associated aortic dissection in a mouse model of Marfan syndrome.

Women with Marfan syndrome (MFS) are at high risk for pregnancy-associated aortic dissection. Pathogenic models that singularly invoke hemodynamic stress are difficult to reconcile with predominant postnatal occurrence of aortic tear, often occurring weeks to months after delivery. In consideration of events that peak at term, are sustained after delivery, and might synergize with previously defined signaling pathways implicated in aneurysm progression, we examined the hormone oxytocin, which initiates uterine contraction and milk letdown for the duration of lactation through phosphorylation of extracellular signal-regulated kinase (ERK). In a mouse model of MFS that shows highly penetrant postnatal aortic dissection, risk was strongly attenuated by preventing lactation or use of an oxytocin receptor antagonist. Survival correlated inversely with the extent of ERK activation in the aortic wall, and strong protection was observed upon attenuation of ERK phosphorylation using an inhibitor of ERK kinase (MEK) or the U.S. Food and Drug Administration-approved medication hydralazine, offering potential therapeutic strategies for pregnancy-associated vascular catastrophe in the setting of MFS.

Oxytocin regulates reunion affiliation with a pairmate following social separation in marmosets.

While separation from significant social partners produces a host of neurobiological and behavioral perturbations, including behavioral distress and increased glucocorticoid production, positive social interactions upon reunion are critical for the reestablishment of normative relationship dynamics and the attenuation of the biobehavioral stress response. The hormone oxytocin has critical and pervasive roles in reproductive and behavioral processes across the lifespan, and plays a particularly prominent role in social bonding. In this study, we examined the extent that oxytocin modulates interactions with a pairmate following separation challenges that varied in both social context (isolation; separation) and duration (long; short), in marmosets. We demonstrated that the impact of pharmacological manipulations of the oxytocin system on the expression of affiliation upon reunion depended on both the context and duration of the separation challenge. Specifically, marmosets treated with an oxytocin antagonist spent less time in proximity with their pairmate upon reunion following a long-separation challenge. During the short-separation challenge, marmosets engaged in more social gaze when separated with an opposite-sex stranger, but not when separated with their mate. Furthermore, marmosets that received the most social gaze from opposite-sex strangers spent the most time in proximity with their long-term mate upon reunion. We also showed that marmosets treated with an OT agonist received increased levels of gaze from opposite-sex strangers, but not from their mate. Overall, these results suggest that marmosets are sensitive to the nature of the social interactions during separation, and subsequently alter their expression of affiliation upon reunion with their long-term mate. These findings further implicate oxytocin as a bond-enhancing molecule that regulates the reestablishment of normative levels of affiliation with a mate following separation, and add to the emerging literature that suggests the OT system underlies critical behavioral processes that contribute to the preservation of long-lasting social bonds.

Intragastric preloads of l-tryptophan reduce ingestive behavior via oxytocinergic neural mechanisms in male mice.

Human and laboratory animal studies suggest that dietary supplementation of a free essential amino acid, l-tryptophan (TRP), reduces food intake. It is unclear whether an acute gastric preload of TRP decreases consumption and whether central mechanisms underlie TRP-driven hypophagia. We examined the effect of TRP administered via intragastric gavage on energy- and palatability-induced feeding in mice. We sought to identify central mechanisms through which TRP suppresses appetite. Effects of TRP on consumption of energy-dense and energy-dilute tastants were established in mice stimulated to eat by energy deprivation or palatability. A conditioned taste aversion (CTA) paradigm was used to assess whether hypophagia is unrelated to sickness. c-Fos immunohistochemistry was employed to detect TRP-induced activation of feeding-related brain sites and of oxytocin (OT) neurons, a crucial component of satiety circuits. Also, expression of OT mRNA was assessed with real-time PCR. The functional importance of OT in mediating TRP-driven hypophagia was substantiated by showing the ability of OT receptor blockade to abolish TRP-induced decrease in feeding. TRP reduced intake of energy-dense standard chow in deprived animals and energy-dense palatable chow in sated mice. Anorexigenic doses of TRP did not cause a CTA. TRP failed to affect intake of palatable yet calorie-dilute or noncaloric solutions (10% sucrose, 4.1% Intralipid or 0.1% saccharin) even for TRP doses that decreased water intake in thirsty mice. Fos analysis revealed that TRP increases activation of several key feeding-related brain areas, especially in the brain stem and hypothalamus. TRP activated hypothalamic OT neurons and increased OT mRNA levels, whereas pretreatment with an OT antagonist abolished TRP-driven hypophagia. We conclude that intragastric TRP decreases food and water intake, and TRP-induced hypophagia is partially mediated via central circuits that encompass OT.

Neuropeptide S Activates Paraventricular Oxytocin Neurons to Induce Anxiolysis.

Neuropeptides, such as neuropeptide S (NPS) and oxytocin (OXT), represent potential options for the treatment of anxiety disorders due to their potent anxiolytic profile. In this study, we aimed to reveal the mechanisms underlying the behavioral action of NPS, and present a chain of evidence that the effects of NPS within the hypothalamic paraventricular nucleus (PVN) are mediated via actions on local OXT neurons in male Wistar rats. First, retrograde studies identified NPS fibers originating in the brainstem locus coeruleus, and projecting to the PVN. FACS identified prominent NPS receptor expression in PVN-OXT neurons. Using genetically encoded calcium indicators, we further demonstrated that NPS reliably induces a transient increase in intracellular Ca2+ concentration in a subpopulation of OXT neurons, an effect mediated by NPS receptor. In addition, intracerebroventricular (i.c.v.) NPS evoked a significant somatodendritic release of OXT within the PVN as assessed by microdialysis in combination with a highly sensitive radioimmunoassay. Finally, we could show that the anxiolytic effect of NPS seen after i.c.v. or intra-PVN infusion requires responsive OXT neurons of the PVN and locally released OXT. Thus, pharmacological blockade of OXT receptors as well as chemogenetic silencing of OXT neurons within the PVN prevented the effect of synthetic NPS. In conclusion, our results indicate a significant role of the OXT system in mediating the effects of NPS on anxiety, and fill an important gap in our understanding of brain neuropeptide interactions in the context of regulation of emotional behavior within the hypothalamus.SIGNIFICANCE STATEMENT Given the rising scientific interest in neuropeptide research in the context of emotional and stress-related behaviors, our findings demonstrate a novel intrahypothalamic mechanism involving paraventricular oxytocin neurons that express the neuropeptide S receptor. These neurons respond with transient Ca2+ increase and somatodendritic oxytocin release following neuropeptide S stimulation. Thereby, oxytocin neurons seem essential for neuropeptide S-induced anxiolysis, as this effect was blocked by pharmacological and chemogenetic inhibition of the oxytocin system.

Feeding response following central administration of mesotocin and arginine-vasotocin receptor agonists in chicks (Gallus gallus).

Mesotocin (MT) and arginine-vasotocin (AVT) are posterior pituitary derived hormones in birds and are homologous to mammalian oxytocin (OT) and vasopressin (VP), respectively. We previously reported that intracerebroventricular (ICV) injection of both MT and AVT inhibit feeding and induce wing-flapping in chicks (Gallus gallus). Because both peptides cause similar effects suggests that they might act via common receptors. However, the specific receptors of MT and AVT which mediate their anorexigenic effect have not been clarified in chicks. Thus, the purpose of the present study was to identify the receptor subtypes involved in MT- and AVT-induced anorexia and behavioral patterns by using several agonists. ICV injection of vasopressin-1 receptor agonist (V1R) (homologous to chicken AVT receptor-2 and -4 [VT2R and VT4R, respectively]), significantly decreased food intake while agonists of vasopressin-2 receptor (V2R) and OT receptor (OTR) (homologues of chicken AVT receptor-1 and MT receptor respectively) had no effect. In addition, V1R agonist induced wing-flapping although this was not affected by V2R or OTR agonists. Since VT2R has not been found in the brain of chicks, the present study suggested that VT4R might be related to the anorexigenic effect and wing-flapping induced by MT and AVT in chicks.

Oxytocinergic manipulations in corticolimbic circuit differentially affect fear acquisition and extinction.

The oxytocinergic system promotes social behavior and reduces anxiety. The significant roles and functional interactions of the medial prefrontal cortex and the amygdala in the regulation of fear provide a unique experimental setting to examine the effects of oxytocin on extinction of fear. In this study we manipulated the oxytocin system at different time points in either the infralimbic region of the medial prefrontal cortex (IL-mPFC), the basolateral amygadala (BLA) or in the central amygdala (CeA). Manipulations of the oxytocin following retrieval of fear in the IL-mPFC resulted in facilitation of subsequent extinction. In contrast, in the BLA, manipulating the oxytocinergic system after the retrieval of fear was associated with contrasting effects; whereas the microinjection of exogenous synthetic oxytocin was associated with impaired extinction, the microinfusion of WAY-267474 facilitated extinction. In contrast, intra-BLA microinfusion of the selective agonist TGOT did not affect freezing. Oxytocin manipulations in the CeA had no effect on subsequent extinction. Contrasting effects were also found when the drugs were injected before conditioning. Whereas oxytocin manipulations in the BLA enhanced fear and impaired extinction, in the CeA the microinfusion of the selective agonists (WAY-267474 and TGOT), but not synthetic oxytocin, resulted in reduced freezing levels. These results show that in the rat, the oxytocinergic system differentially regulated fear and extinction in region and temporal-dependent manners and further join data to show that contrary to the prevailing belief that oxytocin is solely involved in reducing fear, oxytocin can also act as an enhancer of fear responses.

Effects of intracerebroventricularly administered carbetocin on social behavior in Holstein steers.

To shed light on the role of central oxytocin (OXT) in regulating social behavior in cattle, the impact of intracerebroventricularly administered OXT agonist, carbetocin (CBT), on the social behavior of a group of familiar steers was investigated. In the first experiment, we determined the dose response of intracerebroventricularly administered CBT (0.5, 5 or 50 nmol) on plasma cortisol level and behavior using 7 steers aged from 6 to 10 months. Five of the steers were assigned to the second experiment. CBT (50 or 200 nmol/200 µl) in artificial cerebrospinal fluid (aCSF) or aCSF (200 µl) was injected into the third ventricle. Immediately after the injection, the animal and two peers were taken outside to the adjacent paddock. Thirty minutes later, maintenance and social behaviors of the animal were observed for 2 hr. CBT had no effect either on the basal cortisol level or on the maintenance and the abnormal behavior in steers with their movement restricted by a stanchion stall in the first experiment. However, in the same steers with no movement restrictions in the second experiment, CBT facilitated lying, probably because of its sedative effect via OXT receptor activation, which disturbed some aspects of social behavior. These results suggest that central OXT receptor activation might not affect social behavior itself among "familiar members", because the stimulation of the central OXT system by intracerebroventricular administration of CBT did not facilitate social behavior between familiar steers.

Oxytocin and vasopressin agonists and antagonists as research tools and potential therapeutics.

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V(1a), V(1b) and V(2) receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V(1b) receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V(1a) agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V(2) /V(1a) antagonist, conivaptan and the V(2) antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V(1a), V(1b) and V(2) antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences.

Allopregnanolone and induction of endogenous opioid inhibition of oxytocin responses to immune stress in pregnant rats.

In virgin rats, systemic administration of interleukin (IL)-1ß (i.e. to mimic infection), increases oxytocin secretion and the firing rate of oxytocin neurones in the supraoptic nucleus (SON). However, in late pregnancy, stimulated oxytocin secretion is inhibited by an endogenous opioid mechanism, preserving the expanded neurohypophysial oxytocin stores for parturition and minimising the risk of preterm labour. Central levels of the neuroactive metabolite of progesterone, allopregnanolone, increase during pregnancy and allopregnanolone acting on GABA(A) receptors on oxytocin neurones enhances inhibitory transmission. In the present study, we tested whether allopregnanolone induces opioid inhibition of the oxytocin system in response to IL-1ß in late pregnancy. Inhibition of 5α-reductase (an allopregnanolone-synthesising enzyme) with finasteride potentiated IL-1ß-evoked oxytocin secretion in late pregnant rats, whereas allopregnanolone reduced the oxytocin response in virgin rats. IL-1ß increased the number of magnocellular neurones in the SON and paraventricular nucleus (PVN) expressing Fos (an indicator of neuronal activation) in virgin but not pregnant rats. In immunoreactive oxytocin neurones in the SON and PVN, finasteride increased IL-1ß-induced Fos expression in pregnant rats. Conversely, allopregnanolone reduced the number of magnocellular oxytocin neurones activated by IL-1ß in virgin rats. Treatment with naloxone (an opioid antagonist) greatly enhanced the oxytocin response to IL-1ß in pregnancy, and finasteride did not enhance this effect, indicating that allopregnanolone and the endogenous opioid mechanisms do not act independently. Indeed, allopregnanolone induced opioid inhibition over oxytocin responses to IL-1ß in virgin rats. Thus, in late pregnancy, allopregnanolone induces opioid inhibition over magnocellular oxytocin neurones and hence on oxytocin secretion in response to immune challenge. This mechanism will minimise the risk of preterm labour and prevent the depletion of neurohypophysial oxytocin stores, which are required for parturition.

Functional selective oxytocin-derived agonists discriminate between individual G protein family subtypes.

We used a bioluminescence resonance energy transfer biosensor to screen for functional selective ligands of the human oxytocin (OT) receptor. We demonstrated that OT promoted the direct engagement and activation of G(q) and all the G(i/o) subtypes at the OT receptor. Other peptidic analogues, chosen because of specific substitutions in key OT structural/functional residues, all showed biased activation of G protein subtypes. No ligand, except OT, activated G(oA) or G(oB), and, with only one exception, all of the peptides that activated G(q) also activated G(i2) and G(i3) but not G(i1), G(oA), or G(oB), indicating a strong bias toward these subunits. Two peptides (DNalOVT and atosiban) activated only G(i1) or G(i3), failed to recruit ß-arrestins, and did not induce receptor internalization, providing the first clear examples of ligands differentiating individual G(i/o) family members. Both analogs inhibited cell proliferation, showing that a single G(i) subtype-mediated pathway is sufficient to prompt this physiological response. These analogs represent unique tools for examining the contribution of G(i/o) members in complex biological responses and open the way to the development of drugs with peculiar selectivity profiles. This is of particular relevance because OT has been shown to improve symptoms in neurodevelopmental and psychiatric disorders characterized by abnormal social behaviors, such as autism. Functional selective ligands, activating a specific G protein signaling pathway, may possess a higher efficacy and specificity on OT-based therapeutics.

Oxytocin selectively gates fear responses through distinct outputs from the central amygdala.

Central amygdala (CeA) projections to hypothalamic and brain stem nuclei regulate the behavioral and physiological expression of fear, but it is unknown whether these different aspects of the fear response can be separately regulated by the CeA. We combined fluorescent retrograde tracing of CeA projections to nuclei that modulate fear-related freezing or cardiovascular responses with in vitro electrophysiological recordings and with in vivo monitoring of related behavioral and physiological parameters. CeA projections emerged from separate neuronal populations with different electrophysiological characteristics and different response properties to oxytocin. In vivo, oxytocin decreased freezing responses in fear-conditioned rats without affecting the cardiovascular response. Thus, neuropeptidergic signaling can modulate the CeA outputs through separate neuronal circuits and thereby individually steer the various aspects of the fear response.

Postpartum hemorrhage: evidence-based medical interventions for prevention and treatment.

Postpartum hemorrhage (PPH) remains a significant contributor to maternal morbidity and mortality throughout the world. The majority of research on this topic has focused on efforts to prevent PPH. Sound data exist that active management of the third stage of labor can reduce the occurrence of PPH. Although there remains debate regarding the optimal protocol for active management, it appears at this time that oxytocin is the preferable uterotonic to use. Misoprostol may be a reasonable option where parenteral administration of an uterotonic is not feasible. There is little evidence to guide treatment decisions should PPH occur.

Receptor and behavioral pharmacology of WAY-267464, a non-peptide oxytocin receptor agonist.

The widely reported effects of oxytocin (OT) on CNS function has generated considerable interest in the therapeutic potential for targeting this system for a variety of human psychiatric diseases, including anxiety disorders, autism, schizophrenia, and depression. The utility of synthetic OT, as both a research tool and neurotherapeutic, is limited by the physiochemical properties inherent in most neuropeptides, notably its short half-life and poor blood brain barrier penetration. Subsequently, the discovery and development of non-peptide molecules that act as selective agonists of the oxytocin receptor (OTR) has been an important goal of the field. In this study, we report the receptor and behavioral pharmacology of WAY-267464, a first generation small-molecule OTR agonist. WAY-267464 is a high-affinity, potent, and selective (vs. V1a, V2, V1b) agonist of the OTR. In assays measuring both behavioral (four-plate test, elevated zero maze) and autonomic (stress-induced hyperthermia) parameters of the anxiety response, WAY-267464 exhibits an anxiolytic-like profile similar to OT. We have demonstrated that the anxiolytic-like profile of WAY-267464 is mediated through central sites of action. WAY-267464 also significantly reverses disruption in prepulse inhibition of the acoustic startle reflex induced by either MK-801 or amphetamine, similar to the antipsychotic-like effects previously reported for OT. Interestingly, in the mouse tail suspension test, WAY-267464 failed to produce changes in immobility that are seen with OT, raising the question of whether the antidepressant-like activity of OT may be working independently of the OTR. A selective OTR antagonist also failed to block the effects of OT on immobility in the TST. The significance of these findings for shaping the clinical development of OTR agonists is discussed.

Cardiac sympatho-excitatory action of PVN-spinal oxytocin neurones.

A significant proportion of the spinally projecting neurones in the paraventricular nucleus are immunoreactive for oxytocin. Some of these oxytocin neurones terminate on sympathetic preganglionic neurones in the upper thoracic spinal cord, a region from which cardiac sympathetic neurones originate. No studies have so far identified a cardiac action of the supraspinal oxytocin neurones. The present study was designed to test the hypothesis that these oxytocin neurones excite spinal cardiac sympathetic neurones. This was done by measuring heart rate changes in response to intrathecal oxytocin and a selective agonist, and to stimulation of paraventricular neurones before and during blockade of spinal sites with selective antagonists. Rats were anaesthetised with chloralose and urethane (50 mg and 650 mg/kg) and recordings were made of heart rate and blood pressure. Drugs in a volume of 10 microl were applied to the upper thoracic spinal cord via a catheter placed intrathecally with its tip at T2. The paraventricular nucleus was explored with a glass micropipette, placed stereotaxically, and filled with d,l-homocysteic acid (DLH, 200 mM) for exciting neurones and pontamine sky blue for marking the position. Oxytocin (0.002 mM) applied to the spinal cord elicited increases in heart rate (26+/-5 beats per minute). This was mimicked by a highly selective oxytocin agonist. These heart rate increases were blocked selectively by two different oxytocin antagonists but unaffected by a V(1a) vasopressin antagonist. Excitation of sites in dorsal and medial parvocellular sub-nuclei of the paraventricular nucleus elicited increases in heart rate (36+/-3 bpm) which were significantly reduced by oxytocin antagonists but not affected by V(1a) antagonist. Also these induced increases in heart rate were unaffected by vagotomy or i.v. atropine but were abolished by i.v. esmolol. It is concluded that there is a population of paraventricular-spinal oxytocin neurones that excite cardiac sympathetic preganglionic neurones controlling heart rate.

Impact of the Merrifield solid phase method on the design and synthesis of selective agonists and antagonists of oxytocin and vasopressin: a historical perspective.

This tribute to Bruce Merrifield traces the author's fortuitous path in 1964 from Vincent du Vigneaud's laboratory to the laboratory of D. W. Woolley to learn the solid phase method and then to his first faculty position in the Department of Biochemistry, McGill University, Montreal in 1965. It recalls the key roles played from early 1966 to July 1967 by Bruce Merrifield, John Stewart, Arnold Marglin, Herb Takashima, and Vincent du Vigneaud in providing key advice to the author's efforts to use the solid phase method to synthesize oxytocin; while simultaneously the du Vigneaud and Merrifield laboratories were collaborating on the solid phase synthesis of deamino-oxytocin. Both syntheses were published in the same issue of the Journal of American Chemical Society in 1968. Also described is how this breakthrough impacted the author's scientific career: by leading to highly productive collaborative studies, initially with Wilbur H. Sawyer and subsequently with others, on the design and synthesis of selective agonists, antagonists, and radioiodinated ligands for oxytocin and vasopressin receptors. These syntheses were greatly facilitated by the contributions of highly talented graduate students, research technicians, and visiting peptide chemists from Hungary, England, Poland, Bulgaria, and China. Many of these peptides have become very valuable pharmacological tools in studies on the peripheral and central effects of oxytocin and vasopressin: further attesting to the profound impact of the solid phase method as the cornerstone for all the discoveries, which he and his collaborators and coworkers have made over the past 40 years.