Facile method for 3D printing conformally onto uneven surfaces and its application to face masks.

Conventional additive manufacturing processes, where parts are built through layer-wise deposition of material on a horizontal plane, can be limiting when a part must be printed or fit onto uneven surfaces. Such situations will arise with increasing frequency as additive manufacturing application areas such as construction and medical devices continue to grow. In this work, we develop a simple and practical approach to generate toolpaths to print 3D structures onto uneven surfaces conformally. The algorithm uses only conventional planar toolpaths of both the structure to be printed and the substrate to be printed on and converts these to non-planar toolpaths, allowing easy integration with existing additive manufacturing workflows. The technique is demonstrated by printing flexible seals onto bespoke rigid face mask frames conformally via a conventional single-material 3D printer using the generated conformal toolpath. A notable improvement in air seal performance was observed for customized face masks with conformal soft seals compared to conventionally 3D-printed fully rigid face masks. This also shows the potential of the developed toolpath generation method to aid in the prototyping and fabrication of conformal medical and other devices.

Generalisable 3D printing error detection and correction via multi-head neural networks.

Material extrusion is the most widespread additive manufacturing method but its application in end-use products is limited by vulnerability to errors. Humans can detect errors but cannot provide continuous monitoring or real-time correction. Existing automated approaches are not generalisable across different parts, materials, and printing systems. We train a multi-head neural network using images automatically labelled by deviation from optimal printing parameters. The automation of data acquisition and labelling allows the generation of a large and varied extrusion 3D printing dataset, containing 1.2 million images from 192 different parts labelled with printing parameters. The thus trained neural network, alongside a control loop, enables real-time detection and rapid correction of diverse errors that is effective across many different 2D and 3D geometries, materials, printers, toolpaths, and even extrusion methods. We additionally create visualisations of the network's predictions to shed light on how it makes decisions.

Integrative conjugative elements of the ICEPan family play a potential role in Pantoea ananatis ecological diversification and antibiosis.

Pantoea ananatis is a highly versatile enterobacterium isolated from diverse environmental sources. The ecological diversity of this species may be attributed, in part, to the acquisition of mobile genetic elements. One such element is an Integrative and Conjugative Element (ICE). By means of in silico analyses the ICE elements belonging to a novel family, ICEPan, were identified in the genome sequences of five P. ananatis strains and characterized. PCR screening showed that ICEPan is prevalent among P. ananatis strains isolated from different environmental sources and geographic locations. Members of the ICEPan family share a common origin with ICEs of other enterobacteria, as well as conjugative plasmids of Erwinia spp. Aside from core modules for ICEPan integration, maintenance and dissemination, the ICEPan contain extensive non-conserved islands coding for proteins that may contribute toward various phenotypes such as stress response and antibiosis, and the highly diverse ICEPan thus plays a major role in the diversification of P. ananatis. An island is furthermore integrated within an ICEPan DNA repair-encoding locus umuDC and we postulate its role in stress-induced dissemination and/or expression of the genes on this island.

Oxytocinase in the female rat hypothalamus: a novel mechanism controlling oxytocin neurones during lactation.

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase; P-LAP) is expressed both peripherally and centrally. P-LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, although its role in the hypothalamus is unclear. We investigated P-LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, as well as its role in vivo during the milk-ejection reflex. P-LAP mRNA and protein were expressed in magnocellular neurones of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurones co-expressed P-LAP without strong subcellular co-localisation of oxytocin and P-LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P-LAP showed a redistribution of P-LAP to within 1 µm of the plasma membrane in the somata of oxytocin neurones during lactation. Both P-LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c.v. injection of amastatin in anaesthetised suckling mothers increased the frequency of reflex milk ejections. Because hypothalamic P-LAP expression and activity increase in lactation, and the prevention of its action mimics central oxytocin administration, we conclude that P-LAP regulates auto-excitatory oxytocin actions during the suckling-induced milk-ejection reflex.

Diet-induced obesity may affect the uterine immune environment in early-mid pregnancy, reducing NK-cell activity and potentially compromising uterine vascularization.

OBJECTIVES: To investigate the effect of obesity in early-mid pregnancy on crucial pregnancy hormones and the uterine immune environment. BACKGROUND: Obesity impacts reproductive ability, adversely affecting conception and leading to complications in pregnancy. Obesity is often regarded as a stress state and an immune disease, both of which may contribute to pregnancy failure. We previously demonstrated that stress in early pregnancy greatly alters progesterone secretion. As progesterone is an immunomodulator, altered progesterone secretion may adversely modify the maternal immune system. In the current study, we test the hypothesis that obesity during pregnancy adversely alters the uterine immune environment. METHODS: An obese mouse model was created by feeding C57/BL6 mice on a high-fat (HF)/sugar diet for 12 weeks before pregnancy. Control mice were fed on lower-fat/sugar chow. Mice were mated, and on day 7.5 of pregnancy plasma progesterone and prolactin were measured by immunoassay. Cells from the uterus-draining inguinal lymph nodes were collected for analysis of the uterine immune response by flow cytometry. RESULTS: Diet-induced obesity increased the secretion of progesterone and altered a number of uterine natural killer (NK)- and T-cell responses. These included a marked reduction in the percentage of leucocyte-derived NK cells and reduced expression of interferon-γ (IFN-γ) in the NK cells compared with control mice. CONCLUSIONS: Maternal obesity, induced by an HF diet, may lead to a reduction in the expression of IFN-γ in NK cells. NK-cell-derived IFN-γ is reported to be involved in supporting uterine spiral artery remodelling. Thus, obesity in early pregnancy may compromise vascularization by reducing the expression of IFN-γ-positive NK cells. Furthermore, the expression of uterine CD8(+) cells was reduced in the HF diet-fed mice, suggesting obesity may adversely alter the maternal immune adaptation that is essential for effective pregnancy.

Sexual behaviour and neuronal activation in the vomeronasal pathway and hypothalamus of food-deprived male rats.

As feeding and mating are mutually-exclusive goal-orientated behaviours, we investigated whether brief food deprivation would impair the display of sexual behaviour of male rats. Analysis of performance in a sexual incentive motivation test revealed that, similar to fed males, food-deprived males preferred spending time in the vicinity of receptive females rather than nonreceptive females. Despite this, food-deprived males were more likely to be slow to mate than normally-fed males, and a low dose of the satiety peptide α-melanocyte-stimulating-hormone attenuated the effect of hunger. Using Fos immunocytochemistry, we compared neuronal activity in the vomeronasal projection pathway in response to oestrous cues from receptive females between food-deprived and fed males. As in fed males, more Fos expression was seen in the rostral part of the bed nucleus of the stria terminalis and in the medial preoptic area in food-deprived males, confirming that food-deprived males can recognise and respond to female oestrous cues. However, although there was also an increase in Fos expression in the bed nucleus of the accessory tract and in the posteromedial amygdala in fed males, no increases were seen in these areas in food-deprived rats. We also found selective attenuation in the activation of lateral posterior paraventricular nucleus (lpPVN) oxytocin neurones in food-deprived males. Taken together, the data show that, although food-deprived males can still become sexually motivated, copulation is delayed, and this is accompanied by variations in neuronal activity in the vomeronasal projection pathway. We propose that, in hungry rats, the lpPVN oxytocin neurones (which project to the spinal cord and are involved in maintaining penile erection) facilitate the transition from motivation to intromission, and their lack of activation impairs intromission, and thus delays mating.

Differential changes in the hypothalamic-pituitary-adrenal axis and prolactin responses to stress in early pregnant mice.

Stress can cause pregnancy failure but it is unclear how the mother's neuroendocrine system responds to stress to impair mechanisms establishing implantation. We analysed stress-evoked hypothalamic-pituitary-adrenal (HPA) axis responses in early pregnant mice. HPA axis secretory responses to immune stress in early-mid pregnancy were strong and similar to that in virgins, although activation of hypothalamic vasopressin neurones, rather than corticotrophin-releasing hormone neurones, may be more important in the stress response in pregnancy. The site and mode of detrimental glucocorticoid action in pregnancy is not established. Because circulating prolactin is important for progesterone secretion and pregnancy establishment, we also hypothesised that stress negatively impacts on prolactin and its neuroendocrine control systems in early pregnant mice. Basal prolactin secretion was profoundly inhibited by either immune or fasting stress in early pregnancy. Prolactin release is inhibited by tonic dopamine release from tuberoinfundibular (TIDA) neurones. However, immune stress did not increase TIDA neurone activity in the median eminence in pregnant mice [measured by 3,4-dihydroxyphenylacetic acid (DOPAC) content and the DOPAC:dopamine ratio]. By contrast, both immune stress and fasting caused weak induction of Fos in TIDA neurones. However, Fos induction does not always reflect dopamine secretion. Taken together, the data suggest that the stress-evoked profound reduction in prolactin secretion does not involve substantially increased dopamine activity as anticipated. In pregnancy, there was also attenuated recruitment of parvocellular paraventricular nucleus neurones and increased activation of brainstem noradrenergic nuclei after immune stress, indicating that other mechanisms may be involved in the suppression of prolactin secretion. In summary, low prolactin and increased circulating glucocorticoids together may partly explain how a mother's endocrine system mediates stress-induced pregnancy failure.

Effects of cholecystokinin in the supraoptic nucleus and paraventricular nucleus are negatively modulated by leptin in 24-h fasted lean male rats.

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK-8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK-8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK-induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.

Increased sensitivity of monoamine release in the supraoptic nucleus in late pregnancy: region- and stimulus-dependent responses.

Oxytocin neurone activation at birth depends upon noradrenaline-mediated signals from the uterus via a brainstem pathway, as well as on factors within the supraoptic nucleus (SON), including oxytocin itself, and the system adapts during pregnancy to optimise the delivery process. We determined whether noradrenaline release in the SON in response to stimuli activating brainstem inputs or antidromically activating magnocellular neurones is enhanced at term pregnancy. Noradrenaline, serotonin and dopamine concentrations were measured in microdialysis samples collected from the dorsal and ventral SON before, during and after either i.v. cholecystokinin (CCK) or neural stalk stimulation in virgin and late pregnant rats. Each stimulus transiently increased noradrenaline and serotonin but not dopamine concentration in the dorsal SON, and responses were increased on days 21 and 22 of pregnancy compared to day 20 pregnant and virgin rats. Neural stalk stimulation induced sensitisation to subsequent stalk stimulation and so the responses in the dorsal SON were doubled; on day 22 of pregnancy, the area under the curve of monoamine concentration was 3.4-fold greater than in virgins, suggesting that adaptations perinatally enhance responsiveness. In conclusion, there are enhanced responses of noradrenaline and serotonin release in the SON that can generate very high, transient extracellular concentrations at term. This may be a consequence of neuroendocrine adaptations in late pregnancy and probably contributes to optimal oxytocin neurone activation during parturition.

BoLA-DR peptide binding pockets are fundamental for foot-and-mouth disease virus vaccine design in cattle.

This large study investigates the associations between bovine major histocompatibility complex DRB3 alleles and their binding pockets with the immune response to a 40-mer peptide derived from foot-and-mouth disease virus (FMDV) VP1. A crossbred (Charolais and Holstein) cattle population (n=197) was immunised with the FMDV peptide and specific IgG1 and IgG2 responses were measured. Eighteen different DRB3 alleles were detected in this population, with several exhibiting highly significant associations with antibody response. Allele DRB3*1601 was correlated with relatively low IgG1 and IgG2 responses (p<0.001), whereas DRB3*1001 was associated with relatively high IgG1 and IgG2 responses (p<0.001). In contrast the allele *0901 which ranked highest for IgG1 response, only came 14th for IgG2 response. The amino acids at several positions within the peptide binding cleft of the DR molecule showed significant associations (p<0.001) with the level of antibody response. Further analysis showed that specific residues within binding pockets are likely to be crucial to vaccine design. In particular, polymorphisms at position beta70 in pocket 4 were strongly linked to the magnitude of response and highly significant associations were found for position beta57 in pocket 9 and position beta56 in pocket 10. Glutamic acid at position beta70 was associated with low FMDV peptide specific IgG1 and IgG2 response, whereas arginine at beta70 was associated (p<0.001) with a high FMDV peptide specific IgG1 and IgG2 levels. The data indicates that the amino acids within the binding pockets of the DRB3 alleles are critical for determining the degree of immune response and in addition may affect the ratio of IgG1/IgG2, which in turn will influence the efficacy of the peptide to induce protective immunity.

Dopamine-oxytocin interactions in penile erection.

Dopamine and oxytocin have established roles in the central regulation of penile erection in rats; however, the neural circuitries involved in a specific erectile context and the interaction between dopamine and oxytocin mechanisms remain to be elucidated. The medial preoptic area (MPOA), supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus may serve as candidate sites because they contain oxytocin cells, receive dopaminergic inputs and have been implicated in mediating masculine sexual behavior. Double immunofluorescence revealed that substantial numbers of oxytocin cells in the MPOA, SON and PVN possess dopamine D(2), D(3) and D(4) receptors. In anaesthetized rats, using intracavernous pressure as a physiological indicator of erection, blockade of lumbosacral oxytocin receptors (UK, 427843) reduced erectile responses to a nonselective dopamine agonist (apomorphine), suggesting that dopamine recruits a paraventriculospinal oxytocin pathway. In conscious males in the absence of a female, penile erection elicited by a D(2)/D(3) (Quinelorane) but not D(4) (PD168077) agonist was associated with activation of medial parvocellular PVN oxytocin cells. In another experiment where males were given full access to a receptive female, a D(4) (L-745870) but not D(2) or D(3) antagonist (L-741626; nafadotride) inhibited penile erection (intromission), and this was correlated with SON magnocellular oxytocin neuron activation. Together, the data suggest dopamine's effects on hypothalamic oxytocin cells during penile erection are context-specific. Dopamine may act via different parvocellular and magnocellular oxytocin subpopulations to elicit erectile responses, depending upon whether intromission is performed. This study demonstrates the potential existence of interaction between central dopamine and oxytocin pathways during penile erection, with the SON and PVN serving as integrative sites.

Adaptive responses of the maternal hypothalamic-pituitary-adrenal axis during pregnancy and lactation.

Over the past 40 years, it has been recognised that the maternal hypothalamic-pituitary-adrenal (HPA) axis undergoes adaptations through pregnancy and lactation that might contribute to avoidance of adverse effects of stress on the mother and offspring. The extent of the global adaptations in the HPA axis has been revealed and the underlying mechanisms investigated within the last 20 years. Both basal, including the circadian rhythm, and stress-induced adrenocorticotrophic hormone and glucocorticoid secretory patterns are altered. Throughout most of pregnancy, and in lactation, these changes predominantly reflect reduced drive by the corticotropin-releasing factor (CRF) neurones in the parvocellular paraventricular nucleus (pPVN). An accompanying profound attenuation of HPA axis responses to a wide variety of psychological and physical stressors emerges after mid-pregnancy and persists until the end of lactation. Central to this suppression of stress responsiveness is reduced activation of the pPVN CRF neurones. This is consequent on the reduced effectiveness of the stimulation of brainstem afferents to these CRF neurones (for physical stressors) and of altered processing by limbic structures (for emotional stressors). The mechanism of reduced CRF neurone responses to physical stressors in pregnancy is the suppression of noradrenaline release in the PVN by an up-regulated endogenous opioid mechanism, which is induced by neuroactive steroid produced from progesterone. By contrast, in lactation suckling the young provides a neural stimulus that dampens the HPA axis circadian rhythm and reduces stress responses. Reduced noradrenergic input activity is involved in reduced stress responses in lactation, although central prolactin action also appears important. Such adaptations limit the adverse effects of excess glucocorticoid exposure on the foetus(es) and facilitate appropriate metabolic and immune responses.

The identification of potential alternative biomarkers of nitrofurazone abuse in animal derived food products.

Semicarbazide (SEM) was considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone and used as a marker of nitrofurazone abuse. It was recently discovered that SEM can arise in food from sources other than nitrofurazone. This uncertainty over the source of SEM may be overcome if alternative markers specific to tissue-bound nitrofurazone residues can be determined. The structure of nitrofurazone metabolites in vivo and particular proteins to which they are bound are not known. These proteins with altered structure due to the presence of the drug metabolites can be considered as potential alternative biomarkers of nitrofurazone abuse. The proteins implicated in the in vivo binding of nitrofurazone were separated and identified. A crude mixture of proteins extracted from the liver of a rat treated with the drug was separated using a series of different techniques such as preparative isoelectric focusing and size exclusion HPLC. Multiple fractions were assayed by LC-MS/MS to detect the presence of SEM. The proteins containing SEM residues were identified by peptide mass mapping using trypsin digestion and MALDI-TOF. The first protein identified as containing high concentration of SEM was albumin. It was also shown that low molecular weight species within a protein mixture whose main constituent was glutathione S-transferase contained a high concentration of SEM. The chemical composition of these components is under investigation. Preliminary data suggest the SEM forms part of a nitrofurazone metabolite conjugated to glutathione.

Social stress induces hypothalamo-pituitary-adrenal axis responses in lactating rats bred for high trait anxiety.

Hypothalamo-pituitary-adrenal axis responses to various stressors are typically attenuated during lactation, including in rats selectively bred for high or low anxiety. As high-anxiety dams are more aggressive towards intruders than low-anxiety dams during maternal defence, we investigated their hypothalamo-pituitary-adrenal axis responses to this social stress. Maternal defence induced elevated stress responses in high-anxiety dams only; nerve-growth-factor-induced gene B mRNA expression in the parvocellular paraventricular nucleus and adrenocorticotropin hormone secretory responses were substantially enhanced after maternal defence. In contrast, secretory responses to a non-social stress (elevated platform) were not different between high- and low-anxiety dams. Thus, responsiveness of the stress axis in lactation is dependent upon the innate level of anxiety of the dam and, as a consequence, her reactiveness to social threat.

Effects of alpha-melanocyte-stimulating hormone on magnocellular oxytocin neurones and their activation at intromission in male rats.

The peptides alpha-melanocyte-stimulating hormone (alpha-MSH) and oxytocin have very similar effects on several behaviours, including male sexual behaviour. Both induce penile erection and enhance copulatory behaviour when given centrally, suggesting that their central actions are not independent. Here, we used intromission as a physiological stimulus to investigate whether some central effects of alpha-MSH during male sexual behaviour are mediated by oxytocin neurones. We used the expression of the immediate-early gene product Fos to investigate oxytocin neurone activation at intromission and after intracerebroventricular (i.c.v.) administration of alpha-MSH (1 microg/5 microl) and studied the effects of i.c.v. administration of a MC4 receptor antagonist on Fos expression and on the latency of male rats to exhibit sexual behaviour in the presence of a receptive female. In rats that showed intromission, Fos was expressed in magnocellular oxytocin neurones in both the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but there was no significant activation of parvocellular oxytocin neurones of the PVN. Similarly, alpha-MSH increased Fos expression in magnocellular oxytocin neurones but had little or no effect in parvocellular oxytocin neurones. In male rats that achieved intromission, central injection of a MC4 receptor antagonist significantly attenuated the increase in Fos expression in magnocellular oxytocin neurones in both the PVN and the SON and increased mount and intromission latencies compared to vehicle-injected controls. Together, the results indicate that magnocellular oxytocin neurones are involved in the central regulation of male sexual behaviour, and that some of the central effects of alpha-MSH are likely to be mediated by magnocellular oxytocin neurones.

Reduced activity of the noradrenergic system in the paraventricular nucleus at the end of pregnancy: implications for stress hyporesponsiveness.

We investigated whether changes in noradrenaline neurotransmission in the hypothalamus could explain the hyporesponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis in late pregnancy. Noradrenaline release within the hypothalamic paraventricular nucleus in response to swim stress, as estimated by microdialysis and high-performance liquid chromatography, was lower in 20-day pregnant rats compared to virgin rats. Driving a central noradrenergic pathway using intravenous cholecystokinin increased adrenocorticotropic hormone (ACTH) secretion in virgin rats, but the response was significantly less in 16-day and 20-day pregnant rats. Thus, the activity of noradrenergic inputs to the paraventricular nucleus and the HPA axis is attenuated in late pregnancy. The sensitivity of the HPA axis to noradrenaline in pregnancy was investigated by intracerebroventricular administration of an alpha1-receptor antagonist, benoxathian, before and during exposure to swim stress. In virgin rats, benoxathian increased basal and stress-induced ACTH secretion, but in late pregnant rats the benoxathian effects were attenuated, indicating reduced sensitivity of the HPA axis to noradrenaline neurotransmission and/or the inability of the system to become disinhibited at this time. alpha1A-adrenoreceptor mRNA expression in the parvocellular and magnocellular paraventricular nucleus, measured by in situ hybridisation, was decreased in late pregnant compared to virgin rats. Additionally, blocking endogenous opioid inhibition with naloxone pretreatment restored the ACTH secretory response to cholecystokinin in pregnant rats. Thus, in late pregnancy, there is reduced noradrenergic input to the paraventricular nucleus and reduced alpha1A-receptor expression in the paraventricular nucleus, both of which may contribute to the reduced responsiveness of the HPA axis in pregnancy.

No stress response of the hypothalamo-pituitary-adrenal axis in parturient rats: lack of involvement of brain oxytocin.

During parturition, the basal activity of the hypothalamo-pituitary-adrenal (HPA) axis of Wistar rats is strongly attenuated, whereas the oxytocin system is activated. We investigated the secretory responses of the HPA axis and oxytocin to exposure to a mild emotional stressor (airpuff) comparing virgin female, d 22 pregnant, and parturient rats. Furthermore, as the brain oxytocin system is activated in parturition and oxytocin has been shown to inhibit HPA axis responses in virgin rats, the role of brain oxytocin in the regulation of stress responses during parturition was investigated by intracerebroventricular administration of an oxytocin receptor antagonist before stressor exposure (0.75 micro g/5 micro l). In virgin female rats, exposure to airpuff increased ACTH (2.5 +/- 0.34-fold) and corticosterone (5.1 +/- 2.3-fold) secretion, but in late pregnancy and parturition, the stress-induced increase in ACTH (pregnancy: 1.9 +/- 0.41-fold; parturition: 1.3 +/- 0.13-fold) and corticosterone secretion (parturition: 1.8 +/- 0.40-fold) were strongly attenuated. Oxytocin secretion remained unchanged in response to airpuff in both virgin and parturient rats despite higher overall plasma concentrations in the latter. Oxytocin receptor blockade in the brain elevated basal and stress-induced ACTH secretion in virgin but not pregnant or parturient rats and had no effect on oxytocin secretion either in virgin or parturient rats. We conclude that the reactivity of the HPA axis to external stressors is strongly attenuated during parturition, and this cannot be disinhibited by blocking the receptor-mediated action of brain oxytocin.

Beta-endorphin cells in the arcuate nucleus: projections to the supraoptic nucleus and changes in expression during pregnancy and parturition.

Supraoptic nucleus oxytocin neurone activity and secretion are inhibited in late pregnancy and parturition by endogenous opioids. Here, we investigated alterations in the projections and gene expression of beta-endorphin/pro-opiomelanocortin neurones in the arcuate nucleus in the pregnant rat. All regions of the arcuate nucleus were found to contain cells immunoreactive for beta-endorphin fluorescent microbeads retrogradely transported from the supraoptic nucleus, and double-labelled neurones (beta-endorphin plus microbeads), showing that beta-endorphin neurones throughout the arcuate nucleus project to the supraoptic nucleus. There was an increase in the number of beta-endorphin-immunoreactive cells in the arcuate nucleus and an increase in the density of beta-endorphin fibres within the supraoptic nucleus and peri-supraoptic region in late pregnancy and parturition, suggesting enhanced expression of beta-endorphin and increased beta-endorphin innervation of the supraoptic nucleus. Pro-opiomelanocortin mRNA expression in the arcuate nucleus increased in late compared to early pregnancy: the number of positive neurones significantly increased in the caudal region. Fos expression (an indicator of neuronal activation) in the arcuate nucleus was colocalized in beta-endorphin neurones in both proestrus and parturient rats, but the number of positive cells did not increase during parturition, suggesting lack of activation of beta-endorphin neurones at birth. Thus, beta-endorphin cells in the arcuate nucleus project to the supraoptic nucleus and increased innervation during pregnancy may explain the enhanced endogenous opioid inhibition of oxytocin neurones.

The importance of oxytocin mechanisms in the control of mouse parturition.

The role of oxytocin in parturition in mice was investigated. Pup birth profiles, blood samples and brains were collected from parturient mice observed under red light conditions in a reversed light:dark photoperiod. Peripheral administration of an oxytocin antagonist in a dose-dependent manner delayed the birth of subsequent pups, indicating that oxytocin is required for a normal pup birth profile. Oxytocin neurones were activated during birth as shown by both increased immediate early gene ( Fos) expression in oxytocin neurones in the supraoptic nucleus and increased plasma oxytocin concentrations during birth. In addition, the nucleus of the tractus solitarius and the olfactory bulbs, sites that process inputs to oxytocin neurones, become activated during parturition. Exposure to stress during parturition halted subsequent deliveries; at this stage plasma oxytocin concentrations were not higher than those of virgin mice, and birth was restored by administration of oxytocin. Administration of beta-adrenergic antagonist (propranolol) also restored stress-delayed birth, whereas administration of ritrodrine (beta-agonist) delayed birth in non-stressed mice, indicating that adrenergic mechanisms contribute to stress-delayed births in mice. Administration of morphine (mu-opioid agonist) delayed births transiently, but naloxone (opioid antagonist) did not prevent stress-delayed birth, indicating that endogenous opioids do not appear to contribute to neuroendocrine or uterine mechanisms that promote birth in mice. Therefore, despite evidence in oxytocin knockout mice that oxytocin is not essential for parturition in this species, the results of the present study indicate that oxytocin neurone activity and secretion contribute to the birth process in normal mice.