Measuring oxytocin release in response to gavage: Computational modelling and assay validation.

In the present experiments, we tested the conclusion from previous electrophysiological experiments that gavage of sweet food and systemically applied insulin both stimulate oxytocin secretion. To do so, we measured oxytocin secretion from urethane-anaesthetised male rats, and demonstrated a significant increase in secretion in response to gavage of sweetened condensed milk but not isocaloric cream, and a significant increase in response to intravenous injection of insulin. We compared the measurements made in response to sweetened condensed milk with the predictions from a computational model, which we used to predict plasma concentrations of oxytocin from the published electrophysiological responses of oxytocin cells. The prediction from the computational model was very closely aligned to the levels of oxytocin measured in rats in response to gavage.

Provocative tests with Kisspeptin-10 and GnRH set the scene for determining social status and environmental impacts on reproductive capacity in male African lions (Panthera leo).

Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.

Oxytocin-a social peptide? Deconstructing the evidence.

In this paper, we analyse the claim that oxytocin is a 'social neuropeptide'. This claim originated from evidence that oxytocin was instrumental in the initiation of maternal behaviour and it was extended to become the claim that oxytocin has a key role in promoting social interactions between individuals. We begin by considering the structure of the scientific literature on this topic, identifying closely interconnected clusters of papers on particular themes. We then analyse this claim by considering evidence of four types as generated by these clusters: (i) mechanistic studies in animal models, designed to understand the pathways involved in the behavioural effects of centrally administered oxytocin; (ii) evidence from observational studies indicating an association between oxytocin signalling pathways and social behaviour; (iii) evidence from intervention studies, mainly involving intranasal oxytocin administration; and (iv) evidence from translational studies of patients with disorders of social behaviour. We then critically analyse the most highly cited papers in each segment of the evidence; we conclude that, if these represent the best evidence, then the evidence for the claim is weak. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.

The multiple faces of the oxytocin and vasopressin systems in the brain.

Classically, hypothalamic neuroendocrine cells that synthesise oxytocin and vasopressin were categorised in two major cell types: the magnocellular and parvocellular neurones. It was assumed that magnocellular neurones project exclusively to the pituitary gland where they release oxytocin and vasopressin into the systemic circulation. The parvocellular neurones, on the other hand, project within the brain to regulate discrete brain circuitries and behaviours. Within the last few years, it has become evident that the classical view of these projections is outdated. It is now clear that oxytocin and vasopressin in the brain are released extrasynaptically from dendrites and from varicosities in distant axons. The peptides act principally to modulate information transfer through conventional synapses (such as glutamate synapses) by actions at respective receptors that may be preferentially localised to synaptic regions (on either side of the synapse) to alter the 'gain' of conventional synapses.

Identification of peripheral oxytocin-expressing cells using systemically applied cell-type specific adeno-associated viral vector.

Oxytocin is primarily synthesised in the brain and is widely known for its role in lactation and parturition after being released into the blood from the posterior pituitary gland. Nevertheless, peripheral tissues have also been reported to express oxytocin. Using systemic injection of a recombinant adeno-associated virus vector, we investigated the expression of the green fluorescent protein Venus under the control of the oxytocin promoter in the gastrointestinal tract, pancreas and testes of adult rats. Here, we confirm that the vector infects oxytocin neurones of the enteric nervous system in ganglia of the myenteric and submucosal plexuses. Venus was detected in 25%-60% of the ganglia in the myenteric and submucosal plexuses identified by co-staining with the neuronal marker PGP9.5. Oxytocin expression was also detected in the islets of Langerhans in the pancreas and the Leydig cells of the testes. Our data illustrate that peripheral administration of the viral vector represents a powerful method for selectively labelling oxytocin-producing cells outside the brain.

Somato-dendritic vasopressin and oxytocin secretion in endocrine and autonomic regulation.

Somato-dendritic secretion was first demonstrated over 30 years ago. However, although its existence has become widely accepted, the function of somato-dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato-dendritic secretion was demonstrated and are among the neurones for which the functions of somato-dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato-dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra- and inter-population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato-dendritic vasopressin and oxytocin have also been proposed to act as hormone-like signals in the brain. There is some evidence that somato-dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin- or oxytocin-containing axons but, to date, there is no conclusive evidence for, or against, hormone-like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.

Coding of odors in the anterior olfactory nucleus.

Odorant molecules stimulate olfactory receptor neurons, and axons of these neurons project into the main olfactory bulb where they synapse onto mitral and tufted cells. These project to the primary olfactory cortex including the anterior olfactory nucleus (AON), the piriform cortex, amygdala, and the entorhinal cortex. The properties of mitral cells have been investigated extensively, but how odor information is processed in subsequent brain regions is less well known. In the present study, we recorded the electrical activity of AON neurons in anesthetized rats. Most AON cells fired in bursts of 2-10 spikes separated by very short intervals (<20 ms), in a period linked to the respiratory rhythm. Simultaneous recordings from adjacent neurons revealed that the rhythms of adjacent cells, while locked to the same underlying rhythm, showed marked differences in phase. We studied the responses of AON cells to brief high-frequency stimulation of the lateral olfactory tract, mimicking brief activation of mitral cells by odor. In different cells, such stimuli evoked transient or sustained bursts during stimulation or, more commonly, post-stimulation bursts after inhibition during stimulation. This suggests that, in AON cells, phase shifts occur as a result of post-inhibitory rebound firing, following inhibition by mitral cell input, and we discuss how this supports processing of odor information in the olfactory pathway. Cells were tested for their responsiveness to a social odor (the bedding of a strange male) among other simple and complex odors tested. In total, 11 cells responded strongly and repeatedly to bedding odor, and these responses were diverse, including excitation (transient or sustained), inhibition, and activation after odor presentation, indicating that AON neurons respond not only to the type of complex odor but also to temporal features of odor application.

Social creatures: Model animal systems for studying the neuroendocrine mechanisms of social behaviour.

The interaction of animals with conspecifics, termed social behaviour, has a major impact on the survival of many vertebrate species. Neuropeptide hormones modulate the underlying physiology that governs social interactions, and many findings concerning the neuroendocrine mechanisms of social behaviours have been extrapolated from animal models to humans. Neurones expressing neuropeptides show similar distribution patterns within the hypothalamic nucleus, even when evolutionarily distant species are compared. During evolution, hypothalamic neuropeptides and releasing hormones have retained not only their structures, but also their biological functions, including their effects on behaviour. Here, we review the current understanding of the mechanisms of social behaviours in several classes of animals, such as worms, insects and fish, as well as laboratory, wild and domesticated mammals.

Effects of optogenetic stimulation of vasopressinergic retinal afferents on suprachiasmatic neurones.

Physiological circadian rhythms are orchestrated by the hypothalamic suprachiasmatic nucleus (SCN). The activity of SCN cells is synchronised by environmental signals, including light information from retinal ganglion cells (RGCs). We recently described a population of vasopressin-expressing RGCs (VP-RGC) that send axonal projections to the SCN. To determine how these VP-RGCs influence the activity of cells in the SCN, we used optogenetic tools to specifically activate their axon terminals within the SCN. Rats were intravitreally injected with a recombinant adeno-associated virus to express the channelrhodopsin-2 and the red fluorescent protein mCherry under the vasopressin promoter (VP-ChR2mCherry). In vitro recordings in acute brain slices showed that approximately 30% of ventrolateral SCN cells responded to optogenetic stimulation with an increase in firing rate that progressively increased during the first 200 seconds of stimulation and which persisted after the end of stimulation. Finally, application of a vasopressin V1A receptor antagonist dampened the response to optogenetic stimulation. Our data suggest that optogenetic stimulation of VP-RGC axons within the SCN influences the activity of SCN cells in a vasopressin-dependent manner.

The role of vasopressin in olfactory and visual processing.

Neural vasopressin is a potent modulator of behaviour in vertebrates. It acts at both sensory processing regions and within larger regulatory networks to mediate changes in social recognition, affiliation, aggression, communication and other social behaviours. There are multiple populations of vasopressin neurons within the brain, including groups in olfactory and visual processing regions. Some of these vasopressin neurons, such as those in the main and accessory olfactory bulbs, anterior olfactory nucleus, piriform cortex and retina, were recently identified using an enhanced green fluorescent protein-vasopressin (eGFP-VP) transgenic rat. Based on the interconnectivity of vasopressin-producing and sensitive brain areas and in consideration of autocrine, paracrine and neurohormone-like actions associated with somato-dendritic release, we discuss how these different neuronal populations may interact to impact behaviour.

12th World Congress on Neurohypophysial Hormones Mangaratiba, Rio de Janeiro, Brazil - July 26th-29th, 2017.

This Special Issue of Journal of Neuroendocrinology comprises six reviews and eight original research articles describing recent advances in the field of Vasopressin and Oxytocin hormone research based on presentations given at the 12th World Congress on Neurohypophysial Hormones held in July 2017, in Brazil. We, the Chair of the Local Organising Committee and Guest Editor (Maria José A. Rocha) and Guest/Senior Editors for this issue (Celia Sladek and Mike Ludwig), would like to thank the contributors for their excellent reviews and original research articles, the colleagues who reviewed these articles and the Editorial Board of Journal of Neuroendocrinology for their enthusiastic support of the Special Issue. This article is protected by copyright. All rights reserved.

Vasopressin casts light on the suprachiasmatic nucleus.

KEY POINTS: A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light-induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo-entrainment of biological rhythms. ABSTRACT: In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light-evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c-fos in the SCN, which is involved in photic entrainment of circadian rhythms.

Amygdala Kisspeptin Neurons: Putative Mediators of Olfactory Control of the Gonadotropic Axis.

Kisspeptins and their receptors are potent regulators of the gonadotropic axis. Kisspeptin neurons are found mainly in the hypothalamic arcuate nucleus and the anteroventral periventricular nucleus. However, there is also a third population of kisspeptin neurons, located in the amygdala. We used fluorescence immunohistochemistry to quantify and localize the amygdala kisspeptin neurons and to reveal close apposition and putative innervations by vasopressinergic and tyrosine hydroxylase-positive dopaminergic neurons. Using microinjections of retro- and anterograde tracers, and viral transfection systems in rats and transgenic mice, we showed reciprocal connectivity between the accessory olfactory bulb and the amygdala kisspeptin neurons. In vitro recordings indicate an inhibitory action of kisspeptin on mitral cells in the accessory olfactory bulb. Using viral specific-cell gene expression in transgenic mice in combination with double immunofluorescence histochemistry, we found that the amygdala kisspeptin neurons also project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area. Our neuroanatomical and electrophysiological data suggest that amygdala kisspeptin neurons integrate social behaviour and odour information into GnRH neurons in the preoptic area to coordinate the gonadotropic axis and the appropriate output behaviour to odour cues.

The rat suprachiasmatic nucleus: the master clock ticks at 30 Hz.

KEY POINTS: Light-responsive neurones in the rat suprachiasmatic nucleus discharge with a harmonic distribution of interspike intervals, whereas unresponsive neurones seldom do. This harmonic patterning has a fundamental frequency of close to 30 Hz, and is the same in light-on cells as in light-off cells, and is unaffected by exposure to light. Light-on cells are more active than light-off cells in both subjective day and subjective night, and both light-on cells and light-off cells respond more strongly to changes in light intensity during the subjective night than during the subjective day. Paired recordings indicate that the discharge of adjacent light-responsive cells is very tightly synchronized. The gap junction inhibitor carbenoxolone increases the spontaneous activity of suprachiasmatic nucleus neurones but does not block the harmonic discharge patterning. ABSTRACT: The suprachiasmatic nucleus (SCN) of the hypothalamus has an essential role in orchestrating circadian rhythms of behaviour and physiology. In the present study, we recorded from single SCN neurons in urethane-anaesthetized rats, categorized them by the statistical features of their electrical activity and by their responses to light, and examined how activity in the light phase differs from activity in the dark phase. We classified cells as light-on cells or light-off cells according to how their firing rate changed in acute response to light, or as non-responsive cells. In both sets of light-responsive neurons, responses to light were stronger at subjective night than in subjective day. Neuronal firing patterns were analysed by constructing hazard functions from interspike interval data. For most light-responsive cells, the hazard functions showed a multimodal distribution, with a harmonic sequence of modes, indicating that spike activity was driven by an oscillatory input with a fundamental frequency of close to 30 Hz; this harmonic pattern was rarely seen in non-responsive SCN cells. The frequency of the rhythm was the same in light-on cells as in light-off cells, was the same in subjective day as at subjective night, and was unaffected by exposure to light. Paired recordings indicated that the discharge of adjacent light-responsive neurons was very tightly synchronized, consistent with electrical coupling.

Dendritic Release of Neurotransmitters.

Release of neuroactive substances by exocytosis from dendrites is surprisingly widespread and is not confined to a particular class of transmitters: it occurs in multiple brain regions, and includes a range of neuropeptides, classical neurotransmitters, and signaling molecules, such as nitric oxide, carbon monoxide, ATP, and arachidonic acid. This review is focused on hypothalamic neuroendocrine cells that release vasopressin and oxytocin and midbrain neurons that release dopamine. For these two model systems, the stimuli, mechanisms, and physiological functions of dendritic release have been explored in greater detail than is yet available for other neurons and neuroactive substances. © 2017 American Physiological Society. Compr Physiol 7:235-252, 2017.

Intranasal Oxytocin: Myths and Delusions.

Despite widespread reports that intranasal application of oxytocin has a variety of behavioral effects, very little of the huge amounts applied intranasally appears to reach the cerebrospinal fluid. However, peripheral concentrations are increased to supraphysiologic levels, with likely effects on diverse targets including the gastrointestinal tract, heart, and reproductive tract. The wish to believe in the effectiveness of intranasal oxytocin appears to be widespread and needs to be guarded against with scepticism and rigor. Preregistering trials, declaring primary and secondary outcomes in advance, specifying the statistical methods to be applied, and making all data openly available should minimize problems of publication bias and questionable post hoc analyses. Effects of intranasal oxytocin also need proper dose-response studies, and such studies need to include control subjects for peripheral effects, by administering oxytocin peripherally and by blocking peripheral actions with antagonists. Reports in the literature of oxytocin measurements include many that have been made with discredited methodology. Claims that peripheral measurements of oxytocin reflect central release are questionable at best.