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The formation of social memory between individuals of the opposite sex is crucial for expanding mating options or establishing monogamous pair bonding. A specialized neuronal circuit that regulates social memory could enhance an individual's mating opportunities and provide a parallel pathway for computing social behaviors. While the influence of light exposure on various forms of memory, such as fear and object memory, has been studied, its modulation of social recognition memory remains unclear. Here, we demonstrate that acute exposure to light impairs social recognition memory (SRM) in mice. Unlike sound and touch stimuli, light inhibits oxytocin neurons in the supraoptic nucleus (SON) via M1 SON-projecting intrinsically photosensitive retinal ganglion cells (ipRGCs) and GABAergic neurons in the perinuclear zone of the SON (pSON). We further show that optogenetic activation of SON oxytocin neurons using channelrhodopsin is sufficient to enhance SRM performance, even under light conditions. Our findings unveil a dedicated neuronal circuit through which luminance affects SRM, utilizing a non-image-forming visual pathway, distinct from the canonical modulatory role of the oxytocin system., (© 2023 The Authors.)

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., (© 2023 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yaguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Oxytocin is a hormone secreted from definite neuroendocrine neurons located in specific nuclei in the hypothalamus (mainly from paraventricular and supraoptic nuclei), and its main known function is the contraction of uterine and/or mammary gland cells responsible for parturition and breastfeeding. Among the actions of the peripherally secreted oxytocin is the prevention of different degenerative disorders. These actions have been proven in cell culture and in animal models or have been tested in humans based on hypotheses from previous studies. This review presents the knowledge gained from the previous studies, displays the results from oxytocin intervention and/or treatment and proposes that the well described actions of oxytocin might be connected to other numerous, diverse actions of the biomolecule.

The hormone, oxytocin, is synthesised by magnocellular neurones of the supraoptic and paraventricular nuclei and is released from the posterior pituitary gland into the circulation to trigger uterine contractions during parturition. Kisspeptin fibre density increases around the supraoptic nucleus over pregnancy and intracerebroventricular kisspeptin excites oxytocin neurones only in late pregnancy. However, the mechanism of this excitation is unknown. Here, we found that microdialysis administration of kisspeptin into the supraoptic nucleus consistently increased the action potential (spike) firing rate of oxytocin neurones in urethane-anaesthetised late-pregnant rats (gestation day 18-21) but not in non-pregnant rats. Hazard analysis of action potential firing showed that kisspeptin specifically increased the probability of another action potential firing immediately after each action potential (post-spike excitability) in late-pregnant rats. Patch-clamp electrophysiology in hypothalamic slices showed that bath application of kisspeptin did not affect action potential frequency or baseline membrane potential in supraoptic nucleus neurones. Moreover, kisspeptin superfusion did not affect the frequency or amplitude of excitatory postsynaptic currents or inhibitory postsynaptic currents in supraoptic nucleus neurones. Taken together, these studies suggest that kisspeptin directly activates oxytocin neurones in late pregnancy, at least in part, via increased post-spike excitability. KEY POINTS: Oxytocin secretion is triggered by action potential firing in magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei to induce uterine contractions during birth. In late pregnancy, kisspeptin expression increases in rat periventricular nucleus neurones that project to the oxytocin system. Here, we show that intra-supraoptic nucleus administration of kisspeptin increases the action potential firing rate of oxytocin neurones in anaesthetised late-pregnant rats, and that the increased firing rate is associated with increased oxytocin neurone excitability immediately after each action potential. By contrast, kisspeptin superfusion of hypothalamic slices did not affect the activity of supraoptic nucleus neurones or the strength of local synaptic inputs to supraoptic nucleus neurones. Hence, kisspeptin might activate oxytocin neurons in late pregnancy by transiently increasing oxytocin neuron excitability after each action potential., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Oxytocin is secreted into the periphery by magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) to trigger uterine contraction during birth and milk ejection during suckling. Peripheral oxytocin secretion is triggered by action potential firing, which is regulated by afferent input activity and by feedback from oxytocin secreted into the extracellular space from magnocellular neuron somata and dendrites. A prominent input to oxytocin neurons arises from proopiomelanocortin neurons of the hypothalamic arcuate nucleus that secrete an alpha-melanocyte-stimulating hormone (α-MSH), which inhibits oxytocin neuron firing in non-pregnant rats by increasing somato-dendritic oxytocin secretion. However, α-MSH inhibition of oxytocin neuron firing is attenuated in mid-pregnancy and somato-dendritic oxytocin becomes auto-excitatory in late-pregnancy and lactation. Therefore, we hypothesized that attenuated α-MSH inhibition of oxytocin neuron firing marks the beginning of a transition from inhibition to excitation to facilitate peripheral oxytocin secretion for parturition and lactation. Intra-SON microdialysis administration of α-MSH inhibited oxytocin neuron firing rate by 33 ± 9% in non-pregnant rats but increased oxytocin neuron firing rate by 37 ± 12% in late-pregnant rats and by 28 ± 10% in lactating rats. α-MSH-induced somato-dendritic oxytocin secretion measured ex vivo with oxytocin receptor-expressing "sniffer" cells, was of similar amplitude in PVN slices from non-pregnant and lactating rats but longer-lasting in slices from lactating rats. Hence, α-MSH inhibition of oxytocin neuron activity switches to excitation over pregnancy while somato-dendritic oxytocin secretion is maintained, which might enhance oxytocin neuron excitability to facilitate the increased peripheral secretion that is required for normal parturition and milk ejection., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

During parturition and lactation, oxytocin neurones in the supraoptic and paraventricular nuclei fire high-frequency bursts of action potentials that are coordinated across the entire population. Each burst generates a large pulse of oxytocin release into the circulation to induce uterine contraction for parturition and mammary duct contraction for milk ejection. Bursts are stimulated by cervical stretch during parturition and by suckling during lactation. However, the mechanisms by which these stimuli are translated into episodic bursts are poorly understood, as are the mechanisms that coordinate bursts across the oxytocin neurone population. An elegant series of experiments conducted in the 1980s and 1990s used serial paired recordings to show that oxytocin neurones do not act as a syncytium during bursts; rather, they start each burst within a few hundred milliseconds of each other but with no distinct "leaders" or "followers". In addition to afferent noradrenergic inputs that relay the systemic stimuli to oxytocin neurones, bursts depend on somato-dendritic oxytocin release within the hypothalamus. Hence, bursts are considered to be an emergent property of oxytocin neurones that is bootstrapped by appropriate afferent stimulation. Although much progress was made using traditional electrophysiological recordings in head-fixed anaesthetised animals, research has effectively stalled in the last few decades. However, the emergence of new technologies to monitor neuronal activity in freely-behaving animals has reinvigorated efforts to understand the biology underpinning burst firing in oxytocin neurones. Here, we report the use of fibre photometry to monitor the dynamics of milk ejection bursts in the oxytocin neurone population of freely-behaving mice. This approach will shed light on the neural mechanisms that control the oxytocin bursts underpinning parturition and lactation., (© 2021 British Society for Neuroendocrinology.)

Appropriate interactions between astrocytes and oxytocin neurons in the hypothalamo- neurohypophysial system are essential for normal lactation. To further explore the mechanisms underlying astrocytic modulation of oxytocin neuronal activity, we observed astrocytic plasticity in the supraoptic nucleus of lactating rats with intermittent pup-deprivation (PD, 20 h/day) at early (day 1-5) and middle (day 8-12) stages of lactation. PD at both stages decreased suckling duration and litter's body weight gain. They also significantly increased the expression of glial fibrillary acidic protein (GFAP) in Western blots while increased GFAP filaments and the colocalization of GFAP filaments with aquaporin 4 (AQP4) puncta in astrocyte processes surrounding oxytocin neuronal somata in immunohistochemistry in the supraoptic nucleus. Suckling between adjacent milk ejections but not shortly after them decreased molecular association between GFAP and AQP4. In hypothalamic slices from male rats, oxytocin treatment (0.1 nmol/L, 10 min) significantly reduced the length of GFAP filaments and AQP4 puncta in the processes but increased GFAP staining in the somata. These oxytocin effects were blocked by pretreatment of the slices with N-(1,3,4-Thiadiazolyl) nicotinamide (TGN-020, inhibitor of AQP4, 10 µmol/L, 5 min before oxytocin). In addition, inhibition of AQP4 with TGN-020 blocked excitation in oxytocin neurons evoked by prostaglandin E 2 , a downstream signal of oxytocin receptor and mediator of oxytocin-evoked burst firing, in whole-cell patch-clamp recordings. These results indicate that AQP4-associated astrocytic plasticity is essential for normal oxytocin neuronal activity during lactation and that PD-evoked hypogalactia is associated with astrocytic process expansion following increased GFAP and AQP4 expressions., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Aims: Acute restraint stress (RS) has been reported to cause neuronal activation in the supraoptic nucleus of the hypothalamus (SON). The aim of the study was to evaluate the role of SON on autonomic (mean arterial pressure [MAP], heart rate [HR], and tail temperature), neuroendocrine (corticosterone, oxytocin, and vasopressin plasma levels), and behavioral responses to RS., Methods: Guide cannulas were implanted bilaterally in the SON of male Wistar rats for microinjection of the unspecific synaptic blocker cobalt chloride (CoCl2, 1 mM) or vehicle (artificial cerebrospinal fluid, 100 nL). A catheter was introduced into the femoral artery for MAP and HR recording. Rats were subjected to RS, and it was studied the effect of microinjection of CoCl2 or vehicle into the SON on pressor and tachycardic responses, drop in tail temperature, plasma oxytocin, vasopressin, and corticosterone levels, and anxiogenic-like effect induced by RS., Results: SON pretreatment with CoCl2 reduced the RS-induced MAP and HR increase, without affecting the RS-evoked tail temperature decrease. Microinjection of CoCl2 into areas surrounding the SON did not affect RS-induced increase in MAP and HR, reinforcing the idea that SON influences RS-evoked cardiovascular responses. Also, SON pretreatment with CoCl2 reduced RS-induced increase in corticosterone and oxytocin, without affecting vasopressin plasma levels, suggesting its involvement in RS-induced neuroendocrine responses. Finally, the CoCl2 microinjection into SON inhibited the RS-caused delayed anxiogenic-like effect., Conclusion: The results indicate that SON is an important component of the neural pathway that controls autonomic, neuroendocrine, and behavioral responses induced by RS., (© 2019 S. Karger AG, Basel.)

Myocardial infarction (MI) is a leading cause of death worldwide. For those who survive the acute insult, the progressive dilation of the ventricle associated with chronic heart failure is driven by an adverse increase in circulating levels of the antidiuretic hormone, vasopressin, which is secreted from hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) nerve terminals. Although increased vasopressin neuronal activity has been demonstrated in the latter stages of chronic heart failure, we hypothesised that vasopressin neurones become activated immediately following an acute MI. Male Sprague-Dawley rats were anaesthetised and an acute MI was induced by ligation of the left anterior descending coronary artery. After 90 minutes of myocardial ischaemia, brains were collected. Dual-label immunohistochemistry was used to quantify the expression of Fos protein, a marker of neuronal activation, within vasopressin- or oxytocin-labelled neurones of the hypothalamic PVN and SON. Fos protein and tyrosine hydroxylase within the brainstem were also quantified. The results obtained show that the expression of Fos in both vasopressin and oxytocin neurones of the PVN and SON was significantly elevated as soon as 90 minutes post-MI compared to sham rats. Moreover, Fos protein was also elevated in tyrosine hydroxylase neurones in the nucleus tractus solitarius and rostral ventrolateral medulla of MI rats than sham rats. We conclude that magnocellular vasopressin and oxytocin neuronal activation occurs immediately following acute MI, rather than in the later stages of chronic heart failure. Therefore, prompt vasopressin antagonist therapy as an adjunct treatment for acute MI may impede the progression of ventricular dilatation, which remains a key adverse hallmark of chronic heart failure., (© 2019 British Society for Neuroendocrinology.)

Oxytocin (OXT) that effects the nociception process is mainly synthesized and secreted in the hypothalamic supraoptic nucleus (SON). Although the periaqueductal gray (PAG) hardly synthesizes OXT, OXT in PAG also plays a role in pain regulation. The communication investigates whether OXT in the PAG comes from SON to influence pain modulation. RT-PCR was used to analyze OXT mRNA expression and radioimmunoassay to measure OXT concentration. The results showed that (1) pain stimulation enhanced OXT mRNA expression in the SON at 10 min (268.1 ± 39.2%, p < 0.001) and 20 min (135.4±37.9%, p < 0.05) treatment and did not change in the PAG; (2) OXT level increase in SON perfusion liquid during pain stimulation [236.7±22.1% at 10 min (p < 0.001), 223.1±12.4% at 20 min (p<0.001), 56.1 ± 15.7% at 30 min (p < 0.01) and 11.2±14.2% at 40 min] was earlier than that in PAG perfusion liquid [17.8±9.7% at 10 min, 375.6±35.1% at 20 min (p <  0.001), 123.2±17.7% at 30 min (p <  0.001) and 52.7±22.4% at 40 min (p < 0.05)]; (3) SON excitation (L-glutamate sodium microinjection) induced OXT level increase in PAG perfusion liquid in a dose-dependent manner; (4) the bilateral SON cauterization completely controlled and the right SON cauterization partly reversed the pain stimulation induced-OXT concentration increase in PAG perfusion liquid. The data suggested that OXT in PAG came from SON, which might influence the pain process., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca 2+ channels that provide Ca 2+ for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP 2 ) is a phospholipid membrane component that has been previously shown to modulate Ca 2+ channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP 2 modulates these channels, and for the first time show how PIP 2 modulates Ca V channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP 2 depletion via wortmannin (0.5 μmol/L) inhibits Ca 2+ channel currents in OT but not VP neurons. Additionally, it hyperpolarizes voltage-dependent activation of the channels by ~5 mV while leaving the slope of activation unchanged, properties unaffected in VP neurons. We also identified key differences in baseline currents between the cell types, wherein VP whole cell Ca 2+ currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca 2+ channels in OT neurons, which we show to be mostly an effect on N-type Ca 2+ channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca 2+ channel currents. We conclude that PIP 2 is a modulator that enhances current through N-type channels. This has implications for the afterhyperpolarization (AHP) of OT neurons, as previous work from our laboratory demonstrated the AHP is inhibited by wortmannin, and that its primary activation is from intracellular Ca 2+ contributed by N-type channels., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

In the rat supraoptic nucleus, every oxytocin cell projects to the posterior pituitary, and is involved both in reflex milk ejection during lactation and in regulating uterine contractions during parturition. All are also osmosensitive, regulating natriuresis. All are also regulated by signals that control appetite, including the neural and hormonal signals that arise from the gut after food intake and from the sites of energy storage. All are also involved in sexual behaviour, anxiety-related behaviours and social behaviours. The challenge is to understand how a single population of neurones can coherently regulate such a diverse set of functions and adapt to changing physiological states. Their multiple functions arise from complex intrinsic properties that confer sensitivity to a wide range of internal and environmental signals. Many of these properties have a distant evolutionary origin in multifunctional, multisensory neurones of Urbilateria, the hypothesised common ancestor of vertebrates, insects and worms. Their properties allow different patterns of oxytocin release into the circulation from their axon terminals in the posterior pituitary into other brain areas from axonal projections, as well as independent release from their dendrites., (© 2018 British Society for Neuroendocrinology.)

Oxytocin (OT) is a nonapeptide hormone mainly synthesized in the magnocellular neurons of the paraventricular and supraoptic nucleus of the hypothalamus. In the extra-hypothalamic brain areas, OT acts like neurotransmitters and modulators. The physiological functions of OT are multiple. OT participates to the coordination and control of gonadal development and reproduction. OT appears also as an important regulator of social behaviors such as affiliative, parental, and romantic behaviors. Recent evidence suggests other roles for OT such as potent effects on cardiometabolic functions or involvement in stress-related disorders. The growing interest around the clinical role of OT raised the question of the measurement of OT levels and performances of assays., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca 2+ dependent and activated by Ca 2+ influx through voltage-gated Ca 2+ channels. Across central nervous system neurons that generate Ca 2+ -dependent AHPs, the Ca 2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca 2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca 2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 μM Cd 2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 μM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca 2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca 2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca 2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca 2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca 2+ channel blocker abolished the entire AHP, suggesting that additional Ca 2+ sources are involved.

Angiotensin II (ANG) stimulates the release of arginine vasopressin (AVP) from the neurohypophysis through activation of the AT 1 receptor within the brain, although it remains unclear whether AT 1 receptors expressed on AVP-expressing neurons directly mediate this control. We explored the hypothesis that ANG acts through AT 1A receptors expressed directly on AVP-producing cells to regulate AVP secretion. In situ hybridization and transgenic mice demonstrated localization of AVP and AT 1A mRNA in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN), but coexpression of both AVP and AT 1A mRNA was only observed in the SON. Mice harboring a conditional allele for the gene encoding the AT 1A receptor (AT 1A flox ) were then crossed with AVP-Cre mice to generate mice that lack AT 1A in all cells that express the AVP gene (AT 1A AVP-KO ). AT 1A AVP-KO mice exhibited spontaneously increased plasma and serum osmolality but no changes in fluid or salt-intake behaviors, hematocrit, or total body water. AT 1A AVP-KO mice exhibited reduced AVP secretion (estimated by measurement of copeptin) in response to osmotic stimuli such as acute hypertonic saline loading and in response to chronic intracerebroventricular ANG infusion. However, the effects of these receptors on AVP release were masked by complex stimuli such as overnight dehydration and DOCA-salt treatment, which simultaneously induce osmotic, volemic, and pressor stresses. Collectively, these data support the expression of AT 1A in AVP-producing cells of the SON but not the PVN, and a role for AT 1A receptors in these cells in the osmotic regulation of AVP secretion.

In the main olfactory system, odours are registered at the main olfactory epithelium and are then processed at the main olfactory bulb (MOB) and, subsequently, by the anterior olfactory nucleus (AON), the piriform cortex (PC) and the cortical amygdala. Previously, we reported populations of vasopressin neurones in different areas of the rat olfactory system, including the MOB, accessory olfactory bulb (AOB) and the AON and showed that these are involved in the coding of social odour information. Utilising immunohistochemistry and a transgenic rat in which an enhanced green fluorescent protein reporter gene is expressed in vasopressin neurones (eGFP-vasopressin), we now show a population of vasopressin neurones in the PC. The vasopressin neurones are predominantly located in the layer II of the PC and the majority co-express the excitatory transmitter glutamate. Furthermore, there is no sex difference in the number of neurones expressing vasopressin. Electrical stimulation of the lateral olfactory tract leads to a significant increase in the number of Fos-positive nuclei in the PC, MOB, AOB, dorsal AON and supraoptic nucleus (SON). However, there was only a significant increase in Fos expression in vasopressin cells of the PC and SON. Thus, functionally distinct populations of vasopressin cells are implicated in olfactory processing at multiple stages of the olfactory pathway., (© 2017 British Society for Neuroendocrinology.)

Key Points: A growing body of evidence suggests that epithelial Na + channels (ENaCs) in the brain play a significant role in the regulation of blood pressure; however, the brain structures that mediate the effect are not well understood. Because vasopressin (VP) neurons play a pivotal role in coordinating neuroendocrine and autonomic responses to maintain cardiovascular homeostasis, a basic understanding of the regulation and activity of ENaC in VP neurons is of great interest. We show that high dietary salt intake caused an increase in the expression and activity of ENaC which resulted in the steady state depolarization of VP neurons. The results help us understand one of the mechanisms underlying how dietary salt intake affects the activity of VP neurons via ENaC activity., Abstract: All three epithelial Na + channel (ENaC) subunits (α, β and γ) are located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na + leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β- and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaCs, which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Afterhyperpolarizations (AHPs) generated by repetitive action potentials in supraoptic magnocellular neurons regulate repetitive firing and spike frequency adaptation but relatively little is known about PIP 2 's control of these AHPs. We examined how changes in PIP 2 levels affected AHPs, somatic [Ca 2+ ] i , and whole cell Ca 2+ currents. Manipulations of PIP 2 levels affected both medium and slow AHP currents in oxytocin (OT) neurons of the supraoptic nucleus. Manipulations of PIP 2 levels did not modulate AHPs by influencing Ca 2+ release from IP 3 -triggered Ca 2+ stores, suggesting more direct modulation of channels by PIP 2 . PIP 2 depletion reduced spike-evoked Ca 2+ entry and voltage-gated Ca 2+ currents. PIP 2 appears to influence AHPs in OT neurons by reducing Ca 2+ influx during spiking., Abstract: Oxytocin (OT)- and vasopressin (VP)-secreting magnocellular neurons of the supraoptic nucleus (SON) display calcium-dependent afterhyperpolarizations (AHPs) following a train of action potentials that are critical to shaping the firing patterns of these cells. Previous work demonstrated that the lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) enabled the slow AHP component (sAHP) in cortical pyramidal neurons. We investigated whether this phenomenon occurred in OT and VP neurons of the SON. Using whole cell recordings in coronal hypothalamic slices from adult female rats, we demonstrated that inhibition of PIP 2 synthesis with wortmannin robustly blocked both the medium and slow AHP currents (I mAHP and I sAHP ) of OT, but not VP neurons with high affinity. We further tested this by introducing a water-soluble PIP 2 analogue (diC 8 -PIP 2 ) into neurons, which in OT neurons not only prevented wortmannin's inhibitory effect, but slowed rundown of the I mAHP and I sAHP . Inhibition of phospholipase C (PLC) with U73122 did not inhibit either I mAHP or I sAHP in OT neurons, consistent with wortmannin's effects not being due to reducing diacylglycerol (DAG) or IP 3 availability, i.e. PIP 2 modulation of AHPs is not likely to involve downstream Ca 2+ release from inositol 1,4,5-trisphosphate (IP 3 )-triggered Ca 2+ -store release, or channel modulation via DAG and protein kinase C (PKC). We found that wortmannin reduced [Ca 2+ ] i increase induced by spike trains in OT neurons, but had no effect on AHPs evoked by uncaging intracellular Ca 2+ . Finally, wortmannin selectively reduced whole cell Ca 2+ currents in OT neurons while leaving VP neurons unaffected. The results indicate that PIP 2 modulates both the I mAHP and I sAHP in OT neurons, most likely by controlling Ca 2+ entry through voltage-gated Ca 2+ channels opened during spike trains., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K + current (I A ) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR-I A coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited I A in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited I A , contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of I A and enhanced MNC excitability in RVH rats., Abstract: We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K + current (I A ) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR-I A coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch-clamp electrophysiology and real-time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited I A in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1-NR2A-D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited I A and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished I A magnitude and increased firing activity of MNCs from hypertensive rats., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Integrate-and-fire (IF) models can provide close matches to the discharge activity of neurons, but do they oversimplify the biophysical properties of the neurons? A single compartment Hodgkin-Huxley (HH) model of the oxytocin neuron has previously been developed, incorporating biophysical measurements of channel properties obtained in vitro. A simpler modified integrate-and-fire model has also been developed, which can match well the characteristic spike patterning of oxytocin neurons as observed in vivo. Here, we extended the HH model to incorporate synaptic input, to enable us to compare spike activity in the model with experimental data obtained in vivo. We refined the HH model parameters to closely match the data, and then matched the same experimental data with a modified IF model, using an evolutionary algorithm to optimise parameter matching. Finally we compared the properties of the modified HH model with those of the IF model to seek an explanation for differences between spike patterning in vitro and in vivo. We show that, with slight modifications, the original HH model, like the IF model, is able to closely match both the interspike interval (ISI) distributions of oxytocin neurons and the observed variability of spike firing rates in vivo and in vitro. This close match of both models to data depends on the presence of a slow activity-dependent hyperpolarisation (AHP); this is represented in both models and the parameters used in the HH model representation match well with optimal parameters of the IF model found by an evolutionary algorithm. The ability of both models to fit data closely also depends on a shorter hyperpolarising after potential (HAP); this is explicitly represented in the IF model, but in the HH model, it emerges from a combination of several components. The critical elements of this combination are identified.

Oxytocin is a potent anorexigen and is believed to have a role in satiety signaling. We developed rat models to study the activity of oxytocin neurons in response to voluntary consumption or oral gavage of foods using c-Fos immunohistochemistry and in vivo electrophysiology. Using c-Fos expression as an indirect marker of neural activation, we showed that the percentage of magnocellular oxytocin neurons expressing c-Fos increased with voluntary consumption of sweetened condensed milk (SCM). To model the effect of food in the stomach, we gavaged anesthetized rats with SCM. The percentage of supraoptic nucleus and paraventricular nucleus magnocellular oxytocin-immunoreactive neurons expressing c-Fos increased with SCM gavage but not with gastric distention. To further examine the activity of the supraoptic nucleus, we made in vivo electrophysiological recordings from SON neurons, where anesthetized rats were gavaged with SCM or single cream. Pharmacologically identified oxytocin neurons responded to SCM gavage with a linear, proportional, and sustained increase in firing rate, but cream gavage resulted in a transient reduction in firing rate. Blood glucose increased after SCM gavage but not cream gavage. Plasma osmolarity and plasma sodium were unchanged throughout. We show that in response to high-sugar, but not high-fat, food in the stomach, there is an increase in the activity of oxytocin neurons. This does not appear to be a consequence of stomach distention or changes in osmotic pressure. Our data suggest that the presence of specific foods with different macronutrient profiles in the stomach differentially regulates the activity of oxytocin neurons., (Copyright © 2017 Endocrine Society.)

The magnocellular neurosecretory cells (MNCs) of the hypothalamus regulate body fluid balance by releasing the hormones vasopressin (VP) and oxytocin (OT) in an osmolality-dependent manner. Elevations of external osmolality increase MNC firing and hormone release. MNC osmosensitivity is largely due to activation of a mechanosensitive non-selective cation current that responds to osmotically-evoked changes in MNC volume and is mediated by an N-terminal variant of the TRPV1 channel (∆N TRPV1). We report a novel mechanism by which increases in osmolality may modulate ∆N TRPV1-mediated currents and thus influence MNC electrical behaviour. We showed previously that acute elevations of external osmolality activate the enzyme phospholipase C (PLC) in isolated MNCs. We now show that the osmotic activation of PLC has a time course and dose-dependence that is consistent with a role in MNC osmosensitivity and that it contributes to the osmotically-evoked increase in non-selective cation current in MNCs through a protein kinase C-dependent pathway. We furthermore show that the mechanism of osmotic activation of PLC requires an increase in internal Ca 2+ that depends on influx through L-type Ca 2+ channels. Our data therefore suggest that MNCs possess an osmotically-activated Ca 2+ -dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Key Points: Oxytocin release from the posterior pituitary gland stimulates uterine contraction during birth but the central mechanisms that activate oxytocin neurones for birth are not well characterized. We found that that kisspeptin fibre density around oxytocin neurones increases in late-pregnant rats. These kisspeptin fibres originated from hypothalamic periventricular nucleus neurones that upregulated kisspeptin expression in late pregnancy. Oxytocin neurones were excited by central kisspeptin administration in late-pregnant rats but not in non-pregnant rats or early- to mid-pregnant rats. Our results reveal the emergence of a new excitatory kisspeptin projection to the oxytocin system in late pregnancy that might contribute to oxytocin neurone activation for birth., Abstract: The hormone oxytocin promotes uterine contraction during parturition. Oxytocin is synthesized by magnocellular neurones in the hypothalamic supraoptic and paraventricular nuclei and is released into the circulation from the posterior pituitary gland in response to action potential firing. Systemic kisspeptin administration increases oxytocin neurone activity to elevate plasma oxytocin levels. Here, immunohistochemistry revealed that rats on the expected day of parturition (day 21 of gestation) had a higher density of kisspeptin-positive fibres in the perinuclear zone surrounding the supraoptic nucleus (which provides dense glutamatergic and GABAergic innervation to the supraoptic nucleus) than was evident in non-pregnant rats. Retrograde tracing showed the kisspeptin projections to the perinuclear zone originated from the hypothalamic periventricular nucleus. Quantitative RT-PCR showed that kisspeptin receptor mRNA, Kiss1R mRNA, was expressed in the perinuclear zone-supraoptic nucleus and that the relative Kiss1R mRNA expression does not change over the course of pregnancy. Finally, intracerebroventricular administration of kisspeptin increased the firing rate of oxytocin neurones in anaesthetized late-pregnant rats (days 18-21 of gestation) but not in non-pregnant rats, or in early- or mid-pregnant rats. Taken together, these results suggest that kisspeptin expression is upregulated in the periventricular nucleus projection to the perinuclear zone of the supraoptic nucleus towards the end of pregnancy. Hence, this input might activate oxytocin neurones during parturition., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Background: Social recognition underlies social behavior in animals, and patients with psychiatric disorders associated with social deficits show abnormalities in social recognition. Oxytocin is implicated in social behavior and has received attention as an effective treatment for sociobehavioral deficits. Secretin receptor-deficient mice show deficits in social behavior. The relationship between oxytocin and secretin concerning social behavior remains to be determined., Methods: Expression of c-Fos in oxytocin neurons and release of oxytocin from their dendrites after secretin application were investigated. Social recognition was examined after intracerebroventricular or local injection of secretin, oxytocin, or an oxytocin receptor antagonist in rats, oxytocin receptor-deficient mice, and secretin receptor-deficient mice. Electron and light microscopic immunohistochemical analysis was also performed to determine whether oxytocin neurons extend their dendrites into the medial amygdala., Results: Supraoptic oxytocin neurons expressed the secretin receptor. Secretin activated supraoptic oxytocin neurons and facilitated oxytocin release from dendrites. Secretin increased acquisition of social recognition in an oxytocin receptor-dependent manner. Local application of secretin into the supraoptic nucleus facilitated social recognition, and this facilitation was blocked by an oxytocin receptor antagonist injected into, but not outside of, the medial amygdala. In the medial amygdala, dendrite-like thick oxytocin processes were found to extend from the supraoptic nucleus. Furthermore, oxytocin treatment restored deficits of social recognition in secretin receptor-deficient mice., Conclusions: The results of our study demonstrate that secretin-induced dendritic oxytocin release from supraoptic neurons enhances social recognition. The newly defined secretin-oxytocin system may lead to a possible treatment for social deficits., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Melanocortins stimulate the central oxytocin systems that are involved in regulating social behaviours. Alterations in central oxytocin have been linked to neurological disorders such as autism, and melanocortins have been proposed for therapeutic treatment. In the present study, we investigated how systemic administration of melanotan-II (MT-II), a melanocortin agonist, affects oxytocin neuronal activity and secretion in rats. The results obtained show that i.v., but not intranasal, administration of MT-II markedly induced Fos expression in magnocellular neurones of the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and this response was attenuated by prior i.c.v. administration of the melanocortin antagonist, SHU-9119. Electrophysiological recordings from identified magnocellular neurones of the SON showed that i.v. administration of MT-II increased the firing rate in oxytocin neurones but did not trigger somatodendritic oxytocin release within the SON as measured by microdialysis. Our data suggest that, after i.v., but not intranasal, administration of MT-II, the activity of magnocellular neurones of the SON is increased. Because previous studies showed that SON oxytocin neurones are inhibited in response to direct application of melanocortin agonists, the actions of i.v. MT-II are likely to be mediated at least partly indirectly, possibly by activation of inputs from the caudal brainstem, where MT-II also increased Fos expression., (© 2016 British Society for Neuroendocrinology.)

The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis., Significance Statement: Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT., (Copyright © 2016 the authors 0270-6474/16/3611320-11$15.00/0.)

Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons. The eGFP fluorescence intensities in those nuclei and posterior pituitary were markedly increased after chronic salt loading (2% NaCl in drinking water for 5days). ChR2-eGFP was localized mainly in the membrane of AVP-positive MNCs. Whole-cell patch-clamp recordings were performed from single MNCs isolated from the SON of the transgenic rats, and blue light evoked repetitive action potentials. Our work provides for the first time an optogenetic approach to selectively activate AVP neurons in the rat., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The magnocellular neurosecretory cells (MNCs) of the hypothalamus secrete the neurohormones vasopressin and oxytocin. The systemic release of these hormones depends on the rate and pattern of MNC firing and it is therefore important to identify the ion channels that contribute to the electrical behaviour of MNCs. In the present study, we report evidence for the presence of Na(+) -activated K(+) (KN a ) channels in rat MNCs. KN a channels mediate outwardly rectifying K(+) currents activated by the increases in intracellular Na(+) that occur during electrical activity. Although the molecular identity of native KN a channels is unclear, their biophysical properties are consistent with those of expressed Slick (slo 2.1) and Slack (slo 2.2) proteins. Using immunocytochemistry and Western blot experiments, we found that both Slick and Slack proteins are expressed in rat MNCs. Using whole cell voltage clamp techniques on acutely isolated rat MNCs, we found that inhibiting Na(+) influx by the addition of the Na(+) channel blocker tetrodotoxin or the replacement of Na(+) in the external solution with Li(+) caused a significant decrease in sustained outward currents. Furthermore, the evoked outward current density was significantly higher in rat MNCs using patch pipettes containing 60 mm Na(+) than it was when patch pipettes containing 0 mm Na(+) were used. Our data show that functional KN a channels are expressed in rat MNCs. These channels could contribute to the activity-dependent afterhyperpolarisations that have been identified in the MNCs and thereby play a role in the regulation of their electrical behaviour., (© 2016 British Society for Neuroendocrinology.)

Oxytocin (OXT)-containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo-spinal pathway, as well as the hypothalamo-neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT-mRFP1 neurones that expressed Fos-like immunoreactivity (-IR) and OXT-mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT-mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos-IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin-releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT-saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo-neurohypophysial/-spinal OXTergic pathways may be involved in acute nociceptive responses in rats., (© 2016 British Society for Neuroendocrinology.)

Unlabelled: Studying compensatory capacity of the brain is a pressing issue. To resolve it, diversified experiments should be conducted providing an idea of the underlying mechanisms and possibilities of functional restoration., Aim: To determine the effect of unilateral electrocoagulation of supraoptic (SO) and suprachiasmatic (SCH) hypothalamic nuclei on the appearance of the electroretinogram (ERG)., Material and Methods: We conducted chronic experiments on awake rabbits with a total duration of 30 days., Results: The study revealed considerable changes in the total ERG amplitude (reduction) as well as the amplitudes of a- and b-waves. Thus, for the first 15-30 minutes after coagulation the b-wave showed an increase, while the a-wave appeared completely suppressed. Longer postcoagulation periods were associated with gradual, though incomplete, amplitude regain of the latter., Conclusion: The obtained data suggest that the process of ERG recovery involves compensatory mechanisms of the SO and SCH nuclei that enable partial recovery of the neurotransmitter activity of the retina.

Central oxytocin (OT) promotes feeding termination in response to homeostatic challenges, such as excessive stomach distension, salt loading and toxicity. OT has also been proposed to affect feeding reward by decreasing the consumption of palatable carbohydrates and sweet tastants. Because the OT receptor (OTR) is expressed in the nucleus accumbens core (AcbC) and shell (AcbSh), a site regulating diverse aspects of eating behaviour, we investigated whether OT acts there to affect appetite in rats. First, we examined whether direct AcbC and AcbSh OT injections affect hunger- and palatability-driven consumption. We found that only AcbC OT infusions decrease deprivation-induced chow intake and reduce the consumption of palatable sucrose and saccharin solutions in nondeprived animals. These effects were abolished by pretreatment with an OTR antagonist, L-368,899, injected in the same site. AcbC OT at an anorexigenic dose did not induce a conditioned taste aversion, which indicates that AcbC OT-driven anorexia is not caused by sickness/malaise. The appetite-specific effect of AcbC OT is supported by the real-time polymerase chain reaction analysis of OTR mRNA in the AcbC, which revealed that food deprivation elevates OTR mRNA expression, whereas saccharin solution intake decreases OTR transcript levels. We also used c-Fos immunohistochemistry as a marker of neuronal activation and found that AcbC OT injection increases activation of the AcbC itself, as well as of two feeding-related sites: the hypothalamic paraventricular and supraoptic nuclei. Finally, considering the fact that OT plays a significant role in social behaviour, we examined whether offering animals a meal in a social setting would modify their hypophagic response to AcbC OT injections. We found that a social context abolishes the anorexigenic effects of AcbC OT. We conclude that OT acting via the AcbC decreases food intake driven by hunger and reward in rats offered a meal in a nonsocial setting., (© 2016 British Society for Neuroendocrinology.)

Oxytocin neurones of the rat supraoptic nucleus are osmoresponsive and, with all other things being equal, they fire at a mean rate that is proportional to the plasma sodium concentration. However, individual spike times are governed by highly stochastic events, namely the random occurrences of excitatory synaptic inputs, the probability of which is increased by increasing extracellular osmotic pressure. Accordingly, interspike intervals (ISIs) are very irregular. In the present study, we show, by statistical analyses of firing patterns in oxytocin neurones, that the mean firing rate as measured in bins of a few seconds is more regular than expected from the variability of ISIs. This is consistent with an intrinsic activity-dependent negative-feedback mechanism. To test this, we compared observed neuronal firing patterns with firing patterns generated by a leaky integrate-and-fire model neurone, modified to exhibit activity-dependent mechanisms known to be present in oxytocin neurones. The presence of a prolonged afterhyperpolarisation (AHP) was critical for the ability to mimic the observed regularisation of mean firing rate, although we also had to add a depolarising afterpotential (DAP; sometimes called an afterdepolarisation) to the model to match the observed ISI distributions. We tested this model by comparing its behaviour with the behaviour of oxytocin neurones exposed to apamin, a blocker of the medium AHP. Good fits indicate that the medium AHP actively contributes to the firing patterns of oxytocin neurones during non-bursting activity, and that oxytocin neurones generally express a DAP, even though this is usually masked by superposition of a larger AHP., (© 2015 British Society for Neuroendocrinology.)

Background: Magnocellular neurosecretory neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei synthesize vasopressin and oxytocin in response to signals generated by osmoreceptors and baroreceptors and, respectively, by receptors of the nipples and cervix., Methods: We analyzed the literature identifying relevant articles dealing with synaptic inputs of neural afferent pathways to Vasopressin-and Oxytocin-secreting neurons of SON and PVN., Results: This article focuses on the multisynaptic pathways involved in the regulation of Vasopressin and Oxytocin secretion., Conclusion: An updated topographic description of the afferent pathways involved in the regulation of VPergic and OTergic neurons and their synaptic inputs inducing the stimulus-secretion-coupling has been depicted., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Parent-offspring interactions early in life can permanently shape the developmental path of those offspring. Manipulation of maternal care has long been used to alter the early-life environment of infants and impacts their later social behavior, aggression, and physiology. More recently, naturally occurring variation in maternal licking and grooming behavior has been shown to result in differences in social behavior and stress physiology in adult offspring. We have developed a model of natural variation in biparental care in the prairie vole (Microtus ochrogaster) and have demonstrated an association between the amount of early care received and later social behavior. In this study, we investigate the relationship between early life care and later aggression and neuroendocrine responses following chronic social isolation. Male and female offspring were reared by their high-contact (HC) or low-contact (LC) parents, then housed for 4 weeks post-weaning in social isolation. After 4 weeks, half of these offspring underwent an intrasexual aggression test. Brains and plasma were collected to measure corticotropin-releasing hormone (CRH) and vasopressin (AVP) immunoreactivity and plasma corticosterone (CORT). Male offspring of LC parents engaged in more aggressive behavior in the intrasexual aggression test compared to HC males. Female offspring of HC parents had higher plasma CORT levels after chronic social isolation and increases in the number and density of AVP-immunopositive cells in the supraoptic nucleus following an intrasexual aggression test. These findings show that the impact of early life biparental care on behavior and HPA activity following a social stressor is both sex-dependent and early experience-specific., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The classical model of neurovascular coupling (NVC) implies that activity-dependent axonal glutamate release at synapses evokes the production and release of vasoactive signals from both neurons and astrocytes, which dilate arterioles, increasing in turn cerebral blood flow (CBF) to areas with increased metabolic needs. However, whether this model is applicable to brain areas that also use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the rat hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritic release of neuropeptides, including vasopressin (VP), constitutes a key signaling modality influencing neuronal and network activity. Using a multidisciplinary approach, we investigated vasopressin-mediated vascular responses in SON arterioles of hypothalamic brain slices of Wistar or VP-eGFP Wistar rats. Bath-applied VP significantly constricted SON arterioles (Δ-41 ± 7%) via activation of the V1a receptor subtype. Vasoconstrictions were also observed in response to single VP neuronal stimulation (Δ-18 ± 2%), an effect prevented by V1a receptor blockade (V2255), supporting local dendritic VP release as the key signal mediating activity-dependent vasoconstrictions. Conversely, osmotically driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide-mediated vasodilation (Δ19 ± 2%). Activity-dependent vasodilations were followed by a VP-mediated vasoconstriction, which acted to limit the magnitude of the vasodilation and served to reset vascular tone following activity-dependent vasodilation. Together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between nitric oxide and activity-dependent dendritic released VP, in the generation of proper NVC responses., (Copyright © 2015 the authors 0270-6474/15/355330-12$15.00/0.)

Salt loading (SL) and water deprivation (WD) are experimental challenges that are often used to study the osmotic circuitry of the brain. Central to this circuit is the supraoptic nucleus (SON) of the hypothalamus, which is responsible for the biosynthesis of the hormones, arginine vasopressin (AVP) and oxytocin (OXT), and their transport to terminals that reside in the posterior lobe of the pituitary. On osmotic challenge evoked by a change in blood volume or osmolality, the SON undergoes a function-related plasticity that creates an environment that allows for an appropriate hormone response. Here, we have described the impact of SL and WD compared with euhydrated (EU) controls in terms of drinking and eating behavior, body weight, and recorded physiological data including circulating hormone data and plasma and urine osmolality. We have also used microarrays to profile the transcriptome of the SON following SL and remined data from the SON that describes the transcriptome response to WD. From a list of 2,783 commonly regulated transcripts, we selected 20 genes for validation by qPCR. All of the 9 genes that have already been described as expressed or regulated in the SON by osmotic stimuli were confirmed in our models. Of the 11 novel genes, 5 were successfully validated while 6 were false discoveries.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons., (Copyright © 2015 the American Physiological Society.)

Cholinergic activation of the medial septal area (MSA) with carbachol produces thirst, natriuresis, antidiuresis and pressor response. In the brain, hydrogen peroxide (H2O2) modulates autonomic and behavioral responses. In the present study, we investigated the effects of the combination of carbachol and H2O2 injected into the MSA on water intake, renal excretion, cardiovascular responses and the activity of vasopressinergic and oxytocinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Furthermore, the possible modulation of carbachol responses by H2O2 acting through K+ATP channels was also investigated. Male Holtzman rats (280-320 g) with stainless steel cannulas implanted in the MSA were used. The pre-treatment with H2O2 in the MSA reduced carbachol-induced thirst (7.9±1.0, vs. carbachol: 13.2±2.0 ml/60 min), antidiuresis (9.6±0.5, vs. carbachol: 7.0±0.8 ml/120 min,), natriuresis (385±36, vs. carbachol: 528±46 μEq/120 min) and pressor response (33±5, vs. carbachol: 47±3 mmHg). Combining H2O2 and carbachol into the MSA also reduced the number of vasopressinergic neurons expressing c-Fos in the PVN (46.4±11.2, vs. carbachol: 98.5±5.9 c-Fos/AVP cells) and oxytocinergic neurons expressing c-Fos in the PVN (38.5±16.1, vs. carbachol: 75.1±8.5 c-Fos/OT cells) and in the SON (57.8±10.2, vs. carbachol: 102.7±7.4 c-Fos/OT cells). Glibenclamide (K+ATP channel blocker) into the MSA partially reversed H2O2 inhibitory responses. These results suggest that H2O2 acting through K+ATP channels in the MSA attenuates responses induced by cholinergic activation in the same area., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Feeding increases plasma osmolality and ovarian steroids may influence the balance of fluids. Vasopressin (AVP) neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) express estrogen receptor type β (ERβ), but not estrogen receptor type α (ERα). The circumventricular organs express ERα and project efferent fibers to the PVN and SON. Our aim was to assess whether interactions exist between food state-related osmolality changes and the action of estrogen on AVP neuron activity and estrogen receptor expression. We assessed plasma osmolality and AVP levels; fos-coded protein (FOS)- and AVP-immunoreactivity (-IR) and FOS-IR and ERα-IR in the median preoptic nucleus (MnPO) and organ vasculosum lamina terminalis (OVLT) in estrogen-primed and unprimed ovariectomized rats under the provision of ad libitum food, 48h of fasting, and subsequent refeeding with standard chow or sodium-free food. Refeeding with standard chow increased plasma osmolality and AVP as well as the co-expression of FOS-IR/AVP-IR in the PVN and SON. These responses were not altered by estrogen, with the exception of the decreases in FOS-IR/AVP-IR in the lateral PVN. During refeeding, estrogen modulates only a subpopulation of AVP neurons in the lateral PVN. FOS-ERα co-expression in the ventral median preoptic nucleus (vMnPO) was reduced by estrogen and increased after refeeding with standard chow following fasting. It appears that estrogen may indirectly modulate the activity of AVP neurons, which are involved in the mechanism affected by hyperosmolality-induced refeeding after fasting. This indirect action of estrogen can be at least in part via ERα in the vMnPO., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Kisspeptin is the natural ligand of the G protein-coupled receptor -54 and plays a major role in gonadotropin-releasing hormone secretion in the hypothalamus. Kisspeptin-10 is an endogenous derivative of kisspeptin and has 10 -amino acids. Previous studies have demonstrated that central administration of kisspeptin-10 stimulates the secretion of arginine vasopressin (AVP) in male rats. We examined the effects of kisspeptin-10 on- excitatory synaptic inputs to magnocellular neurosecretory cells (MNCs) including AVP neurons in the supraoptic nucleus (SON) by obtaining in vitro whole-cell patch-clamp recordings from slice preparations of the rat brain. The application of kisspeptin-10 (100 nM-1 μM) significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The kisspeptin-10-induced potentiation of the mEPSCs was significantly attenuated by previous exposure to the kisspeptin receptor antagonist kisspeptin-234 (100 nM) and to the protein kinase C inhibitor bisindolylmaleimide I (20 nM). These results suggest that kisspeptin-10 participates in the regulation of synaptic inputs to the MNCs in the SON by interacting with the kisspeptin receptor., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Magnocellular neurosecretory cells (MNCs) in the rat supraoptic nucleus display clustered firing during hyperosmolality or dehydration. This response is beneficial because this type of activity potentiates vasopressin secretion from axon terminals in the neurohypophysis and thus promotes homoeostatic water reabsorption from the kidney. However, the mechanisms which lead to the generation of clustering activity in MNCs remain unknown. Previous work has shown that clustered firing can be induced in these neurons through the pharmacological activation of NMDA receptors (NMDARs) and that silent pauses observed during this activity are mediated by apamin-sensitive calcium activated potassium (SK) channels. However, it remains unknown if clustered firing can be induced in situ by endogenous glutamate release from axon terminals. Here we show that electrical stimulation of glutamatergic osmosensory afferents in the organum vasculosum lamina terminalis (OVLT) can promote clustering in MNCs via NMDARs and apamin-sensitive channels.We also show that the rate of spike clustering induced by NMDA varies as a bell-shaped function of voltage, and that partial inhibition of SK channels can increase cluster duration and reduce the rate of clustering. Finally,we show that MNCs express neurotensin type 2 receptors, and that activation of these receptors can simultaneously depolarize MNCs and suppress clustered firing induced by bath application of NMDA or by repetitive stimulation of glutamate afferents. These studies reveal that spike clustering can be induced in MNCs by glutamate release from afferent nerve terminals and that that this type of activity can be fine-tuned by neuromodulators such as neurotensin.

The magnocellular neurosecretory cells of the hypothalamus (MNCs) synthesize and secrete vasopressin or oxytocin. A stretch-inactivated cation current mediated by TRPV1 channels rapidly transduces increases in external osmolality into a depolarization of the MNCs leading to an increase in action potential firing and thus hormone release. Prolonged increases in external osmolality, however, trigger a reversible structural and functional adaptation that may enable the MNCs to sustain high levels of hormone release. One poorly understood aspect of this adaptation is somatic hypertrophy. We demonstrate that hypertrophy can be evoked in acutely isolated rat MNCs by exposure to hypertonic solutions lasting tens of minutes. Osmotically evoked hypertrophy requires activation of the stretch-inactivated cation channel, action potential firing, and the influx of Ca(2+). Hypertrophy is prevented by pretreatment with a cell-permeant inhibitor of exocytotic fusion and is associated with an increase in total membrane capacitance. Recovery is disrupted by an inhibitor of dynamin function, suggesting that it requires endocytosis. We also demonstrate that hypertonic solutions cause a decrease in phosphatidylinositol 4,5-bisphosphate in the plasma membranes of MNCs that is prevented by an inhibitor of phospholipase C (PLC). Inhibitors of PLC or protein kinase C (PKC) prevent osmotically evoked hypertrophy, and treatment with a PKC-activating phorbol ester can elicit hypertrophy in the absence of changes in osmolality. These studies suggest that increases in osmolality cause fusion of internal membranes with the plasma membrane of the MNCs and that this process is mediated by activity-dependent activation of PLC and PKC., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The magnocellular vasopressin (VP) and oxytocin (OT) neurones undergo long-term synaptic plasticity to accommodate prolonged hormone demand. By contrast, rapidly induced,transient synaptic plasticity in response to brief stimuli could enable the activation of magnocellular neurones in response to acute challenges. Here, we report a robust short-term potentiation of asynchronous GABAergic synaptic inputs (STP(GABA)) to VP and OT neurones of the hypothalamic supraoptic nucleus elicited by repetitive extracellular electrical stimulation.The STP(GABA) required extracellular Ca2+, but did not require activation of glutamate, VP or OT receptors or nitric oxide synthesis. Presynaptic action potential generation was necessary for the induction, but not the maintenance, of STP(GABA). The STP(GABA) led to a minutes-long GABA(A)receptor-dependent increase in spike frequency in VP neurones, but not in OT neurones,consistent with an excitatory function of GABA in only VP neurones and with the generation of prolonged bursts of action potentials in VP neurones. Therefore, this short-term plasticity of GABAergic synaptic inputs is likely to play very different roles in the regulation of OT and VP neurones and their distinct patterns of physiological activation.

The blockade of central nitric oxide (NO) signaling modifies the thermoregulatory and metabolic adjustments that occur during exercise, thereby impairing physical performance. However, the brain areas involved in this response remain unknown. Nitrergic neurons are present in the hypothalamic areas that are activated during exercise and participate in autonomic and neuroendocrine responses, such as, the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). To investigate whether brain NO signaling affects thermoregulation during exercise through the activation of hypothalamic neurons, rats underwent acute submaximal treadmill exercise (18 mmin(-1), 5% inclination) until fatigue received an intracerebroventricular injection of 1.43 μmol Nω-nitro-l-arginine metil ester (L-NAME), a nitric oxide synthase inhibitor, or saline (SAL). Skin tail temperature (Tsk) and internal body temperature (Ti) were continuously recorded and c-Fos expression was determined in the PVN and the SON. L-NAME treatment reduced physical performance by 48%, which was positively correlated with tail vasodilation capacity, which was reduced by 28%, and negatively correlated with heat storage rate (HSR), which was increased by 38%. Physical exercise until fatigue increased the number of c-Fos-immunoreactive (ir) neurons in the PVN and the SON. L-NAME-treatment significantly reduced the exercise-induced c-Fos expression in the PVN, whereas it had no effect in the SON. Interestingly, the number of c-Fos-ir neurons in the PVN was closely correlated with physical performance and inversely associated with HSR. Thus, the inhibition of central NO attenuates neuronal activation induced by exercise in the PVN, impairs the autonomic regulation of heat dissipation, and anticipates the fatigue. Brain NO seems to play a role in exercise performance through the regulation of neuronal activation in the PVN, but not in the SON, although the SON neurons are also activated by running exercise. Moreover, this role in performance mediated by neuronal activation in the PVN can be related with the improvement of thermoregulatory adjustments that occur during exercise., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present experiments were undertaken to examine whether oxytocin cells in the paraventricular nucleus receive synaptic inputs from the contralateral supraoptic or paraventricular nucleus. Using urethane-anesthetized lactating rats, extracellular action potentials were recorded from single oxytocin or vasopressin cells in the paraventricular nucleus. Electrical stimulation was applied to the contralateral supraoptic nucleus or paraventricular nucleus, and responses of oxytocin or vasopressin cells were analyzed by peri-stimulus time histogram or by change in firing rate of oxytocin or vasopressin cells. Electrical stimulation of the contralateral supraoptic nucleus or paraventricular nucleus did not cause antidromic excitation in oxytocin or vasopressin cells but caused orthodromic responses. Although analysis by peri-stimulus time histogram showed that electrical stimulation of the contralateral supraoptic nucleus or paraventricular nucleus caused orthodromic excitation in both oxytocin and vasopressin cells, the proportion of excited oxytocin cells was greater than that of vasopressin cells. Train stimulation applied to the contralateral supraoptic nucleus or paraventricular nucleus at 10 Hz increased firing rates of oxytocin cells and decreased those of vasopressin cells. The results of the present experiments suggest that oxytocin cells in the paraventricular nucleus receive mainly excitatory synaptic inputs from the contralateral supraoptic nucleus and paraventricular nucleus. Receipt these synaptic inputs to oxytocin cells may contribute to the synchronized activation of oxytocin cells during the milk ejection reflex., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Female and male rats are often described as having a promiscuous mating strategy, yet simple Pavlovian conditioning paradigms, in which a neutral odor or strain-related cues are paired with preferred sexual reward states during an animal's first sexual experiences, shift this strategy toward copulatory and mate preferences for partners bearing the familiar odor or strain cue. We examined whether female rats given exclusive rewarding copulation with one particular male would display mate-guarding behavior, a strong index of monogamous mating. Ovariectomized, hormone-primed female Long-Evans rats were given their first 10 paced sexual experiences at 4-day intervals with a particular unscented male of the same strain. A final test was conducted in an open field 4-days later in which the primed, partnered female was given access to the male partner and a fully-primed competitor female. In this situation, the partnered females mounted the competitor female repeatedly if she came near the vicinity of the male. This behavior prevented the male from copulating with the competitor, and was not displayed if partnered females could not pace the rate of copulatory behavior efficiently during the training trials, nor was it displayed by the competitor females. Fos expression was examined in both the partnered and competitor females after the final open field test. Partnered females had significantly higher expression within the supraoptic nucleus and nucleus accumbens shell compared to partnered females that did not develop this behavior or competitor females. These data show that females engaged in paced copulation with the same male display mate-guarding when exposed to that male and a competitor female. Increased activation of the SON and NAc may underlie this behavior., (Copyright © 2014 Elsevier Inc. All rights reserved.)

It has been shown that the endocannabinoid system is involved in the neurohypophyseal hormone secretion produced by exposure to several different stimuli; however, the influence of this system on neuroendocrine responses during lactation is unclear. Therefore, the aim of our study was to investigate the influence of an acute peripheral administration of WIN55,212-2 (cannabinoid receptor agonist) on behavioral and neuroendocrine responses during lactation. On day 6 of lactation, female rats were treated with vehicle or WIN55,212-2 30 min before the start of our experiments. To evaluate maternal behavior, the pups were returned to their home cages to the side of the cage opposite the previous nest, and the resulting behavior of the lactating rats was recorded for the next 30 min. Aggressive behavior was evaluated for 10 min following the placement of an intruder male rat in the home cage. The plasma level of oxytocin and the amount of milk consumption by the pups were evaluated 15 min after the onset of suckling. In addition, double-labelled c-Fos/oxytocin neurons in the medial magnocellular subdivision of the paraventricular nucleus and in the supraoptic nucleus were quantified for each lactating rat. The results show that WIN decreased maternal care, decreased aggressive behaviors, suppressed maternal anxiolysis, decreased plasma oxytocin levels and milk consumption by pups and decreased activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the changes in the behavioral responses of lactating rats treated with WIN maybe can be related to disruption in the neuroendocrine control of oxytocin secretion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.

The effects of gastrin-releasing peptide (GRP) on the circadian clock in the suprachiasmatic nucleus (SCN) are dependent on the activation of N-methyl-d-aspartate (NMDA) receptors in the SCN. In this study, the interaction between GRP, glutamate and serotonin in the regulation of circadian phase in Syrian hamsters was evaluated. Microinjection of GRP into the third ventricle induced c-fos and p-ERK expression throughout the SCN. Coadministration of an NMDA antagonist or 8-hydroxy-2-di-n-propylamino-tetralin [a serotonin (5-HT)1A,7 agonist, DPAT] with GRP limited c-fos expression in the SCN to a region dorsal to GRP cell bodies. Similar to the effects of NMDA antagonists, DPAT attenuated GRP-induced phase shifts in the early night, suggesting that the actions of serotonin on the photic phase shifting mechanism occur downstream from retinorecipient cells. c-fos and p-ERK immunoreactivity in the supraoptic (SON) and paraventricular hypothalamic nuclei also increased following ventricular microinjection of GRP. Because of this finding, a second set of experiments was designed to test a potential role for the SON in the regulation of clock function. Syrian hamsters were given microinjections of GRP into the peri-SON during the early night. GRP-induced c-fos activity in the SCN was similar to that following ventricular administration of GRP. GRP or bicuculline (a γ-aminobutyric acidA antagonist) administered near the SON during the early night elicited phase delays of circadian activity rhythms. These data suggest that GRP-induced phase-resetting is dependent on levels of glutamatergic and serotonergic neurotransmission in the SCN and implicate activity in the SON as a potential regulator of photic signaling in the SCN., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)