Oxytocin receptor antagonist reverses the blunting effect of pair bonding on fear learning in monogamous prairie voles.

Social relationships among spouses, family members, and friends are known to affect physical and mental health. In particular, long-lasting bonds between socio-sexual partners have profound effects on cognitive, social, emotional, and physical well-being. We have previously reported that pair bonding in monogamous prairie voles (Microtus ochrogaster) is prevented by a single prolonged stress (SPS) paradigm, which causes behavioral and endocrine symptoms resembling post-traumatic stress disorder (PTSD) patients in rats (Arai et al., 2016). Since fear memory function is crucial for anxiety-related disorders such as PTSD, we investigated the effects of pair bonding on fear learning in prairie voles. We applied an SPS paradigm to male prairie voles after the cohabitation with a male (cage-mate group) or female (pair-bonded group). The cage-mate group, but not the pair-bonded group, showed enhanced fear response in a contextual fear conditioning test following the SPS treatment. Immunohistochemical analyses revealed that cFos-positive cells in the central amygdala were increased in the pair-bonded group after the contextual fear conditioning test and that oxytocin immunoreactivity in the paraventricular nucleus of the hypothalamus was significantly higher in the pair-bonded group than the cage-mate group. This pair-bonding dependent blunting of fear memory response was confirmed by a passive avoidance test, another fear-based learning test. Interestingly, intracerebroventricular injection of an oxytocin receptor antagonist 30 min before the passive avoidance test blocked the blunting effect of pair bonding on fear learning. Thus, pair bonding between socio-sexual partners results in social buffering in the absence of the partner, blunting fear learning, which may be mediated by oxytocin signaling.

New Approach to Calculation of Renal Cation Clearance in Rats after V1a Receptor Stimulation.

V1a receptor agonist [Phe2, Ile3, Orn8]-vasopressin produces maximum diuretic and saluretic effects in a dose of 0.1 nmol/100 g body weight: renal excretion of Na+ increases by 50 times in comparison with the initial level over 90 min, excretion of K+, Mg2+, and Са2+ increases by 3, 2, and 16 times, respectively. New formulas were proposed for calculation of the clearance considering total cation content in the extracellular fluid and the time of total effect of the injected dose. After injection of V1a receptors agonist, clearance of Na+ was 7% of its total content in the extracellular fluid, the corresponding values for K+, Mg2+, and Ca2+ were 63, 35, and 6%, respectively. The proposed approach differs significantly from the standard cation excretion evaluation and characterizes selective shifts in ion balance induced by physiologically active substances and possible side effects due to unbalanced loss of certain ions.

Oxytocin-like receptors mediate pair bonding in a socially monogamous songbird.

Although many species form socially monogamous pair bonds, relevant neural mechanisms have been described for only a single species, the prairie vole (Microtus ochrogaster). In this species, pair bonding is strongly dependent upon the nonapeptides oxytocin (OT) and vasopressin, in females and males, respectively. Because monogamy has evolved many times in multiple lineages, data from additional species are required to determine whether similar peptide mechanisms modulate bonding when monogamy evolves independently. Here we test the hypothesis that OT-like receptor activation is required for pair bond formation in the socially monogamous zebra finch (Taeniopygia guttata). Males and females were administered chronic intracerebroventricular infusions of saline or an OT receptor antagonist and were observed twice daily for 3 days in a colony environment. A variety of affiliative, aggressive and other behaviours were quantified. The antagonist produced significant and selective effects on pair bonding (latency to pair; number of sessions paired; stable pairing) and the associated behaviour of allopreening. Importantly, findings for males follow the trends of females; this yields main effects of treatment in two-way ANOVAs, although within-sex analyses are significant only for females. These data provide evidence for both convergent evolution and species diversity in the neuroendocrine mechanisms of pair bonding.

Sex-specific activity and function of hypothalamic nonapeptide neurons during nest-building in zebra finches.

Vertebrate species from fish to humans engage in a complex set of preparatory behaviors referred to as nesting; yet despite its phylogenetic ubiquity, the physiological and neural mechanisms that underlie nesting are not well known. We here test the hypothesis that nesting behavior is influenced by the vasopressin-oxytocin (VP-OT) peptides, based upon the roles they play in parental behavior in mammals. We quantified nesting behavior in male and female zebra finches following both peripheral and central administrations of OT and V1a receptor (OTR and V1aR, respectively) antagonists. Peripheral injections of the OTR antagonist profoundly reduce nesting behavior in females, but not males, whereas comparable injections of V1aR antagonist produce relatively modest effects in both sexes. However, central antagonist infusions produce no effects on nesting, and OTR antagonist injections into the breast produce significantly weaker effects than those into the inguinal area, suggesting that antagonist effects are mediated peripherally, likely via the oviduct. Finally, immunocytochemistry was used to quantify nesting-induced Fos activation of nonapeptide neurons in the paraventricular and supraoptic nuclei of the hypothalamus and the medial bed nucleus of the stria terminalis. Nest-building induced Fos expression within paraventricular VP neurons of females but not males. Because the avian forms of OT (Ile(8)-OT; mesotocin) and VP (Ile(3)-VP; vasotocin) exhibit high affinity for the avian OTR, and because both peptide forms modulate uterine contractility, we hypothesize that nesting-related stimuli induce peptide release from paraventricular vasotocin neurons, which then promote female nesting via peripheral feedback from OTR binding in the oviduct uterus.

Oxytocin-induced analgesia and scratching are mediated by the vasopressin-1A receptor in the mouse.

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Role of spinal V1a receptors in regulation of arterial pressure during acute and chronic osmotic stress.

Vasopressinergic neurons in the paraventricular nucleus project to areas in the spinal cord from which sympathetic nerves originate. This pathway is hypothesized to be involved in the regulation of mean arterial pressure (MAP), particularly under various conditions of osmotic stress. Several studies measuring sympathetic nerve activity support this hypothesis. However, the evidence that spinal vasopressin influences MAP under physiological or pathophysiological conditions in conscious animals is limited. The purpose of this study was to investigate, in conscious rats, if the increases in MAP during acute or chronic osmotic stimuli are due to activation of spinal vasopressin (V1a) receptors. Three conditions of osmotic stress were examined: acute intravenous hypertonic saline, 24- and 48-h water deprivation, and 4 wk of DOCA-salt treatment. Rats were chronically instrumented with an indwelling catheter for intrathecal injections and a radiotelemeter to measure MAP. In normotensive rats, intrathecal vasopressin and V1a agonist increased MAP, heart rate, and motor activity; these responses were blocked by pretreatment with an intrathecal V1a receptor antagonist. However, when the intrathecal V1a antagonist was given during the three conditions of osmotic stress to investigate the role of "endogenous" vasopressin, the antagonist had no effect on MAP, heart rate, or motor activity. Contrary to the hypothesis suggested by previous studies, these findings indicate that spinal V1a receptors are not required for elevations of MAP under conditions of acute or chronic osmotic stress in conscious rats.

Selective blockade of oxytocin and vasopressin V(1a) receptors in anaesthetised rats: evidence that activation of oxytocin receptors rather than V(1a) receptors increases sodium excretion.

BACKGROUND: Although it is known that moderate-to-high doses of the neurohypophysial hormones oxytocin and vasopressin are natriuretic, doubts remain over the identity of the receptors responsible. To address this issue, we have used highly selective antagonists of oxytocin and vasopressin receptors in animals with elevated endogenous circulating levels of the 2 hormones. METHODS: Rats were anaesthetised and prepared surgically for clearance studies, thereby raising plasma oxytocin and vasopressin concentrations. Sodium excretion, glomerular filtration rate and lithium clearance (an index of end-proximal fluid delivery) were measured: first during a control period, then after administration of the selective oxytocin receptor antagonist desGly-NH(2),d(CH(2))(5)[D-Trp(2),Thr(4),Dap(5)]OVT, the selective vasopressin V(1a) receptor antagonist d(CH(2))(5)[Tyr(Me)(2),Dab(5)]AVP, or vehicle alone. RESULTS: Absolute and fractional sodium excretion fell in rats given the oxytocin antagonist (by 32 and 27%, respectively, compared with corresponding values in vehicle-infused rats), but not in those given the V(1a) antagonist or vehicle. Antinatriuresis was associated with a small reduction in the ratio of sodium clearance to lithium clearance (an index of the fraction of distally delivered sodium that escapes reabsorption in the distal nephron). CONCLUSIONS: These results corroborate previous studies showing that activation of oxytocin receptors increases sodium excretion and imply that the natriuretic effect of elevated plasma vasopressin concentrations results from stimulation of oxytocin receptors.

Oxytocin-induced postinhibitory rebound firing facilitates bursting activity in oxytocin neurons.

During parturition and lactation, neurosecretory oxytocin (OT) neurons in the hypothalamus achieve pulsatile hormone secretion by coordinated bursts of firing that occur throughout the neuronal population. This activity is partly controlled by somatodendritic release of OT, which facilitates the onset and recurrence of synchronized bursting. To further investigate the cellular mechanisms underlying the control exerted by OT on the activity of its own neurons, we studied the effects of the peptide on membrane potential and synaptic activity in OT neurons in hypothalamic organotypic slice cultures. Bath application of low concentrations of OT (<100 nM) facilitated GABA(A) receptor-mediated inhibitory transmission through a presynaptic mechanism without affecting membrane potential and excitatory glutamatergic synaptic activity. The facilitatory action of OT on GABAergic transmission was dose-dependent, starting at 25 nM and disappearing at concentrations >100 nM. As shown previously, higher concentrations of OT (>500 nM) had the opposite effect, inhibiting GABA(A) receptors via a postsynaptic mechanism. Surprisingly, OT-mediated facilitation of GABAergic transmission promoted action potential firing in 40% of the neurons. Each action potential occurred at the end of the repolarizing phase of an inhibitory potential. Pharmacological dissection revealed that this firing involved the activation of low-threshold activated calcium channels. Detailed statistical analysis showed that OT-mediated firing upregulated bursting activity in OT neurons. It is thus likely to optimize OT secretion and, as a consequence, facilitate delivery and milk ejection in mammals.

Central oxytocin modulation of acute stress-induced cardiovascular responses after myocardial infarction in the rat.

The present study was aimed at determining the role of centrally released oxytocin in regulation of blood pressure and heart rate (HR) under resting conditions and during an acute air-jet stress in rats with a myocardial infarction and controls infarcted. Four weeks after ligation of a coronary artery or sham surgery, conscious Sprague Dawley rats were subjected to one of the following intracerebroventricular (ICV) infusions: (1) 0.9% NaCl (control), (2) oxytocin, (3) oxytocin receptor antagonist {desGly-NH(2)-d(CH(2))(5)[D-Tyr(2)Thr(4)]OVT}(OXYANT). Resting arterial blood pressure and HR were not affected by any of the ICV infusions either in the infarcted or sham-operated rats. In the control experiments, the pressor and tachycardic responses to the air jet of infarcted rats were significantly greater than in the sham-operated rats. OXYANT significantly enhanced the cardiovascular responses to stress only in the sham-operated rats whereas oxytocin significantly attenuated both responses in the infarcted but not in the sham-operated rats. The results suggest that centrally released endogenous oxytocin significantly reduces the cardiovascular responses to the acute stressor in control rats. This buffering function of the brain-oxytocin system is not efficient during the post-myocardial infarction state, however it may be restored by central administration of exogenous oxytocin.

Central oxytocin alters cortisol and behavioral responses of guinea pig pups during isolation in a novel environment.

The neuropeptide oxytocin plays key roles in social bonding and stress reduction, and thus appears to be a likely mediator of maternal buffering of infant stress responses. In the guinea pig, the presence of the mother in a threatening environment buffers cortisol elevations as well as active (vocalizing) and passive (e.g. crouching) responses typical of isolation in this species; yet, effects of OT in guinea pig pups under any conditions have not been reported. Here, we examined the ability of intracerebroventricular (ICV) OT to moderate plasma cortisol levels and behavior in guinea pig pups isolated in a brightly lit, novel environment, and the ability of a highly selective OT antagonist (OTA) to reduce buffering by the mother. We found that ICV OT moderated cortisol levels and vocalizations, but increased time spent in the crouched stance, particularly in females. In addition, OT modulated other ongoing behaviors in a sex-dependent fashion. In females, OT reduced duration of walking and rearing, and increased time spent quiet, while in males OT increased duration of rearing. OTA, however, was without effect on cortisol levels or behavior. These findings, including sex differences in response, extend results from other species to the guinea pig. Further, while demonstrating that exogenous OT is sufficient to reduce biobehavioral stress responses typical of isolated guinea pig infants, the results suggest that endogenous OT is not necessary for maternal buffering of infant responses in this species.

Oxytocin receptor activity in the ventrocaudal periaqueductal gray modulates anxiety-related behavior in postpartum rats.

Postpartum rats are less anxious than diestrous virgin females, a phenomenon requiring that mothers have recent contact with their infants. Oxytocin (OT) is one of many neurochemicals released intracerebrally while mothers interact with infants, and we investigated whether OT receptor activity in the ventrocaudal periaqueductal gray (cPAGv) contributes to mothers' reduced anxiety. Infusion of the highly specific OT receptor antagonist, desGly-NH2,d(CH2)5[D-Tyr2,Thr-sup-4]OVT, into the cPAGv reduced the percentage of time dams spent in the open arms of an elevated plus-maze, but had no effect on the open-arm behavior of diestrous virgins. Conversely, after separating dams from their litters for 4 hr to increase anxiety, a lower (2 ng) but not higher (5 ng) dose of OT infused into each hemisphere of the cPAGv doubled the percentage of time dams spent in open arms, but did not do so in virgins. OTergic manipulations inconsistently affected risk-assessment behaviors (stretch-attend postures, head dips) in both virgins and dams. Therefore, OT receptor activation in the cPAGv is an important consequence of contact with infants that reduces some anxiety-related behaviors in mother rats.

The role of the vasopressin V1A receptor in oxytocin modulation of methamphetamine primed reinstatement.

The neuropeptide oxytocin has shown promise as an effective therapy in pre-clinical models of methamphetamine (METH) addiction. The nucleus accumbens core (NAcc) has been identified as an important site for oxytocin to inhibit METH behaviours, although previous findings suggest that the effects of oxytocin in the NAcc are mediated by receptors other than the oxytocin receptor (OTR). Oxytocin has high affinity for the vasopressin V1A receptor (V1AR) which has been implicated in numerous oxytocin-dependent social behaviours. The aim of this study was to investigate the involvement of the V1AR in mediating the effect of oxytocin treatment to reduce METH-primed reinstatement of METH-seeking behaviour. Male rats were trained to self-administer intravenous infusions of METH by lever press during daily 2-h fixed ratio 1 scheduled sessions for 20 days. Following extinction of lever pressing, rats were tested for the effects of oxytocin alone, oxytocin co-administered with a selective V1AR antagonist, or oxytocin co-administered with a selective OTR antagonist, on METH-primed reinstatement, when administered systemically, or when microinjected into the NAcc. Systemic administration of oxytocin prevented METH-primed reinstatement, an effect which was significantly reduced by systemic pre-treatment with a V1AR but not OTR antagonist. Local administration of oxytocin into the NAcc reduced METH-primed reinstatement, but not when the V1AR was blocked. Our results demonstrate a substantial role for the V1AR in mediating the inhibitory effects of oxytocin on METH-primed reinstatement, and indicate the need for investigations into the differential involvement of V1ARs and OTRs in oxytocin-induced reduction of METH-related behaviours.

Oxytocin action at the lactotroph is required for prolactin surges in cervically stimulated ovariectomized rats.

Cervical stimulation induces two daily rhythmic prolactin surges, nocturnal and diurnal, which persist for several days. We have shown that a bolus injection of oxytocin initiates a similar prolactin rhythm, which persists despite low levels of oxytocin after injection. This suggests that oxytocin may trigger the cervical stimulation-induced rhythmic prolactin surges. To investigate this hypothesis, we infused an oxytocin antagonist that does not cross the blood-brain barrier for 24 h before and after cervical stimulation and measured serum prolactin. We also measured dopaminergic neuronal activity because mathematical modeling predicted that this activity would be low in the presence of the oxytocin antagonist. We thus tested this hypothesis by measuring dopaminergic neuronal activity in the tuberoinfundibular, periventricular hypophyseal, and tuberohypophyseal dopaminergic neurons. Infusion of oxytocin antagonist before cervical stimulation abolished prolactin surges, and infusion of oxytocin antagonist after cervical stimulation abolished the diurnal and significantly decreased the nocturnal surges of prolactin. The rhythmic prolactin surges returned after the clearance of the oxytocin antagonist. Hypothalamic dopaminergic activity was elevated in antiphase with prolactin surges, and the antiphase elevation was abolished by the oxytocin antagonist in the tuberoinfundibular and tuberohypophyseal dopaminergic neurons, consistent with the mathematical model. These findings suggest that oxytocin is a physiologically relevant prolactin-releasing factor. However, the cervical stimulation-induced prolactin surges are maintained even in the absence of oxytocin actions at the lactotroph, which strongly suggests the maintenance of prolactin surges are not dependent upon oxytocin actions at the pituitary gland.

Neonatal oxytocin manipulations have long-lasting, sexually dimorphic effects on vasopressin receptors.

Developmental exposure to oxytocin (OT) or oxytocin antagonists (OTAs) has been shown to cause long-lasting and often sexually dimorphic effects on social behaviors in prairie voles (Microtus ochrogaster). Because regulation of social behavior in monogamous mammals involves central receptors for OT, arginine vasopressin (AVP), and dopamine, we examined the hypothesis that the long-lasting, developmental effects of exposure to neonatal OT or OTA might reflect changes in the expression of receptors for these peptides. On postnatal day 1, prairie voles were injected intraperitoneally with either OT (1 mg/kg), an OTA (0.1 mg/kg), saline vehicle, or were handled only. At approximately 60 days of age, vasopressin V1a receptors, OT receptors (OTR) and dopamine D2 receptor binding were quantified using receptor autoradiography in brain tissue taken from males and females. Significant treatment effects on V1a binding were found in the bed nucleus of the stria terminalis (BNST), cingulate cortex (CgCtx), mediodorsal thalamus (MdThal), medial preoptic area of the hypothalamus (MPOA), and lateral septum (LS). The CgCtx, MPOA, ventral pallidum, and LS also showed significant sex by treatment interactions on V1a binding. No significant treatment or sex differences were observed for D2 receptor binding. No significant treatment difference was observed for OTR receptor binding, and only a marginal sex difference. Changes in the neuropeptide receptor expression, especially the V1a receptor, may help to explain sexually dimorphic changes in behavior that follow comparable neonatal manipulations.

Involvement of the oxytocin system in the bed nucleus of the stria terminalis in the sex-specific regulation of social recognition.

Sex differences in the oxytocin (OT) system in the brain may explain why OT often regulates social behaviors in sex-specific ways. However, a link between sex differences in the OT system and sex-specific regulation of social behavior has not been tested. Here, we determined whether sex differences in the OT receptor (OTR) or in OT release in the posterior bed nucleus of the stria terminalis (pBNST) mediates sex-specific regulation of social recognition in rats. We recently showed that, compared to female rats, male rats have a three-fold higher OTR binding density in the pBNST, a sexually dimorphic area implicated in the regulation of social behaviors. We now demonstrate that OTR antagonist (5 ng/0.5 µl/side) administration into the pBNST impairs social recognition in both sexes, while OT (100 pg/0.5 µl/side) administration into the pBNST prolongs the duration of social recognition in males only. These effects seem specific to social recognition, as neither treatment altered total social investigation time in either sex. Moreover, baseline OT release in the pBNST, as measured with in vivo microdialysis, did not differ between the sexes. However, males showed higher OT release in the pBNST during social recognition compared to females. These findings suggest a sex-specific role of the OT system in the pBNST in the regulation of social recognition.

Oxytocin receptor subtypes in the pregnant rat myometrium and decidua: pharmacological differentiations.

Oxytocin (OT) has a dual action in the uterus: a uterotonic action on myometrial cells and a prostaglandin (PG)-releasing action on endometrial/decidual cells. It had not been determined whether the OT-binding sites or receptors on the myometrial and the endometrial/decidual membranes are of the same type or may represent two subtypes. Our studies presented in this paper show that isolated day 19-22 pregnant rat uterine horns and myometrial tissues (uterine horns with decidual tissues removed) incubated in Kreb's buffer at 37 C released PGF2 alpha in sustained quantities into the bathing medium. OT stimulated PG release over the basal release rate in a dose-dependent manner in the whole uterine horn but not in the myometrial tissue. Two OT antagonists, P[Phe(Me)2,Thr4]ornithine vasotocin (antagonist A) and desGly-NH2(9),d(CH2)5(1)[Tyr(Me)2,Thr4]ornithine vasotocin (antagonist B) were found to have different effects on the PG-releasing action of OT. At antiuterotonic doses, antagonist A had no antagonism of the PG-releasing action of OT. On the contrary, antagonist A was found to stimulate uterine PG release. Antagonist B was a full OT antagonist. At equivalent antiuterotonic doses, antagonist B inhibited both the uterotonic action and the PG-releasing action of OT. These findings suggest that OT-sensitive PGs are synthesized/released principally in the endometrium/decidua. The myometrial uterotonic OT receptors and the endometrial/decidual PG-releasing OT receptors are two distinct subtypes and can be differentiated. The existence of two OT receptor subtypes in the uterus has important implications in the clinical application of OT antagonists as tocolytics for preterm labor. To be efficacious, OT antagonist therapy needs to block both the uterotonic and the PG-releasing action of OT.

Effects of activation of vasopressin-V1-receptors on regional kidney blood flow and glomerular arteriole diameters.

OBJECTIVES: We tested whether vasoconstriction of juxtamedullary glomerular arterioles contributes to vasopressin V1 -receptor-mediated reductions in medullary perfusion (MBF). DESIGN AND METHODS: The left kidney of pentobarbitone anaesthetized rabbits was denervated, a perivascular flow probe placed around the renal artery and laser-Doppler flow probes positioned in the inner medulla and on the cortical surface. Rabbits then received a 30 min intravenous infusion of [Phe2,Ile3,Orn8]vasopressin (V1 -AG; 30 ng/kg per min; n = 7) or its vehicle (n = 7). Kidneys were perfusion fixed at the final recorded mean arterial pressure (MAP) and filled with methacrylate casting material. Diameters of afferent and efferent arterioles were determined by scanning electron microscopy. RESULTS: V1 -AG increased MAP (19 +/- 3%) and reduced MBF (30 +/- 8%) but not cortical perfusion or total renal blood flow. Vehicle-treatment did not significantly affect these variables. After vehicle- and V1-AG-treatment, juxtamedullary afferent arteriole luminal diameter averaged 15.35 +/- 1.31 and 15.88 +/- 1.86 microm, respectively (P= 0.92), while juxtamedullary efferent arteriole luminal diameter averaged 17.75 +/- 1.86 and 18.36 +/- 2.24 microm, respectively (P= 0.93). CONCLUSIONS: V1-AG reduced MBF but did not significantly affect juxtamedullary arteriolar diameter. Our results therefore do not support a role for juxtamedullary arterioles in producing V1-receptor-mediated reductions in MBF, suggesting that downstream vascular elements (e.g. outer medullary descending vasa recta) might be involved.

Oxytocin antagonists delay the initiation of parturition and prolong its active phase in rats.

The physiological importance of oxytocin for the initiation and maintenance of labour and delivery is controversial. We investigated the effects of two novel peptide oxytocin antagonists on the onset and the progress of delivery in rats implanted with a jugular vein cannula one day before term. During delivery rats were given either an oxytocin antagonist (OVT16, n = 10, or F382, n = 7, 30 micrograms/kg) or vehicle (n = 10, 9) after the birth of the second pup and the time to deliver five more pups was recorded. Other rats were given an injection of F382 (30 micrograms/kg, n = 7) or vehicle (n = 9) after the birth of the fourth pup and the time to deliver three more pups was recorded. In another experiment rats were given repeated injections of F382 (30 or 60 micrograms/kg, n = 13, 11) or vehicle (n = 32) prepartum on the day of expected term and the time of onset and the progress of delivery was recorded. Rats given an antagonist after the second pup delivered the next five pups in 100 +/- 8 min (F382) and 83 +/- 12 min (OVT16), significantly slower than the respective controls (51 +/- 6 and 49 +/- 6 min, U-test, P < 0.05). Four of the 7 rats given F382 after the fourth pup showed no prolongation of delivery (time between pups 4-7: 24.7 +/- 2.9 vs 27.5 +/- 3.1 min in controls), while in the other three rats delivery was prolonged (time between pups 4-7: 86 +/- 4.3 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterisation of the biological effects of neurohypophysial peptides on seminiferous tubules.

Oxytocin (OT) is present in the mammalian testis and has been postulated to play a role in modulation of seminiferous tubule contractility. However, recent evidence suggests that the myoid cells responsible for such contractile activity do not express OT receptors. In this study computer-assisted analysis and time-lapse videomicrography were used to investigate the biological effects of neurohypophysial peptides and their analogues on seminiferous tubule contractility. Adult rat testes were placed in fresh oxygenated Dulbecco's modified Eagle's medium (DMEM) F12 medium, decapsulated and the tubules gently teased apart. A small section of tubule was placed in a microslide chamber and perifused with medium. Seminiferous tubules were treated with OT (2 nM), [Arg8]-vasopressin (AVP, 0.2 nM) or [Thr4,Gly7]-OT (TGOT, 2 nM, 8 nM and 0.2 microM). Specific antagonists were also given simultaneously with OT and AVP treatments. Data were analysed to give arbitrary units of contractility. Both OT and AVP increased tubule contractility, with AVP being at least 10 times more potent than OT. Treatment with the selective OT antagonist, desGly-NH2,d(CH2)5[d-Tyr2,Thr4]-ornithine vasotocin (OTA, 0.2 microM and 2 microM) significantly reduced OT-induced increases in seminiferous tubule contractility but had no effect on AVP-induced responses. In contrast, the AVP antagonist, Phaa-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (AVPA) was more potent at reducing AVP-induced increases than OT-induced responses. The selective non-peptide AVPA SR 49059 blocked the response to both peptides in a similar manner, whilst the non-peptide OTA L367,773 did not block OT-induced increases in seminiferous tubule contractility at doses that were slightly inhibitory to AVP-induced responses. The specific OT agonist TGOT did not induce a contractile response. The data in this study demonstrate that in the testis AVP acts via V1a receptors to stimulate contractile activity and suggest that OT may act via a receptor which differs from the classical V1a and uterine-type OT receptor. These findings support a role for OT in the regulation of seminiferous tubule contractility and raise the possibility that AVP may also be important in this process.

Vasopressin-induction of the immediate early gene, NGFI-A, in cultured hippocampal glial cells.

Our earlier autoradiographic work had documented a wide distribution of vasopressin receptors in the hippocampus [R.E. Brinton, K.W. Gee, J.K. Wamsley, T.P. Davis, H.I. Yamamura, Regional distribution of putative vasopressin receptors in rat brain and pituitary by quantitative autoradiography, in: Proc. Natl. Acad. Sci. USA, 81 (1984) pp. 7248-7252; C. Chen, R.D. Brinton, T.J. Shors, R.F. Thompson, [Arg 8]-Vasopressin-induction of long lasting potentiation of synaptic transmission in the dentate gyrus, Hippocampus 3 (1993) 193-203.] which suggested the possibility that receptors for vasopressin were present in both neurons and glia. In the periphery, vasopressin is a potent mitogen in select proliferative cell types [E. Rozengurt, A. Legg, P. Pettican, Vasopressin stimulation of mouse 3T3 cell growth, Proc. Natl. Acad. Sci. USA, 76 (1979) pp. 1284-1287.] which also suggested a possible association between vasopressin receptor activation and the proliferative capacity of astrocytes. We therefore investigated whether vasopressin would induce the expression of the immediate early response gene, NGFI-A (also known as zif/268, ZENK, egr-1, krox 24), which is associated with initiation of mitogenesis [M. Sheng, M.E. Greenberg, The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) pp. 477-485.]. Cultured hippocampal glial cells were exposed to vasopressin or a selective V1 vasopressin receptor agonist and in situ hybridization for NGFI-A mRNA was conducted. Results of these experiments demonstrated that vasopressin induced a highly significant dose-dependent increase in the number of cells expressing NGFI-A. Studies to determine the receptor subtype mediating vasopressin induction of NGFI-A were conducted utilizing the specific V1 agonist, [Phe2, Ile3, Orn8]-vasopressin. The V1 receptor agonist induced a highly significant dose dependent increase in the number of grains per NGFI-A positive cell. Time course analysis demonstrated that V1 agonist induction of NGFI-A occurred within 5 min, was maximally induced at 15 min of exposure and exhibited a gradual decline within 30 min of exposure which continued to decline over the 60 min time course. Glial cell responsivity was selective in that vasopressin and V1 agonist induction of NGFI-A occurred in a subpopulation of glial cells. Within a sea of glial cells, vasopressin and V1 agonist would induce islands of NGFI-A positive cells. Results of combined immunocytochemical labeling for the astrocyte specific marker, GFAP, and in situ hybridization for NGFI-A demonstrated that V1 agonist-induced NGFI-A expression occurred in GFAP positive cells. We observed no evidence for V1 agonist induction of NGFI-A in neurons. Collectively, these data document that vasopressin, acting via V1 vasopressin receptors, induces a highly significant increase in NGFI-A expression in select GFAP positive hippocampal astrocytes. To our knowledge, these data are the first report of a vasopressin mediated response in hippocampal glial cells. The potential functional significance of these findings is discussed.