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.

Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice.

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.

Oxytocin: A Multi-Functional Biomolecule with Potential Actions in Dysfunctional Conditions; From Animal Studies and Beyond.

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.

α-Melanocyte-stimulating hormone inhibition of oxytocin neurons switches to excitation in late pregnancy and lactation.

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.

Local kisspeptin excitation of rat oxytocin neurones in late pregnancy.

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.

Visualising oxytocin neurone activity in vivo: The key to unlocking central regulation of parturition and lactation.

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.

Astrocytic Modulation of Supraoptic Oxytocin Neuronal Activity in Rat Dams with Pup-Deprivation at Different Stages of Lactation.

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 E2, 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.

The Supraoptic Nucleus of the Hypothalamus Modulates Autonomic, Neuroendocrine, and Behavioral Responses to Acute Restraint Stress in Rats.

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.

Acute myocardial infarction activates magnocellular vasopressin and oxytocin neurones.

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.

Oxytocin in the periaqueductal gray mainly comes form the hypothalamic supraoptic nucleus to participate in pain modulation.

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 ±â€¯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 ±â€¯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.

PIP2 alters of Ca2+ currents in acutely dissociated supraoptic oxytocin neurons.

Magnocellular neurosecretory cells (MNCs) occupying the supraoptic nucleus (SON) contain voltage-gated Ca2+ channels that provide Ca2+ for triggering vesicle release, initiating signaling pathways, and activating channels, such as the potassium channels underlying the afterhyperpolarization (AHP). Phosphotidylinositol 4,5-bisphosphate (PIP2 ) is a phospholipid membrane component that has been previously shown to modulate Ca2+ channels, including in the SON in our previous work. In this study, we further investigated the ways in which PIP2 modulates these channels, and for the first time show how PIP2 modulates CaV channel currents in native membranes. Using whole cell patch clamp of genetically labeled dissociated neurons, we demonstrate that PIP2 depletion via wortmannin (0.5 µmol/L) inhibits Ca2+ 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 Ca2+ currents display more inactivation and shorter deactivation time constants. Wortmannin accelerates inactivation of Ca2+ channels in OT neurons, which we show to be mostly an effect on N-type Ca2+ channels. Finally, we demonstrate that wortmannin prevents prepulse-induced facilitation of peak Ca2+ channel currents. We conclude that PIP2 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 Ca2+ contributed by N-type channels.

The osmoresponsiveness of oxytocin and vasopressin neurones: Mechanisms, allostasis and evolution.

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.

Oxytocin testing and reproductive health: Status and clinical applications.

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.

Specificity in the interaction of high-voltage-activated Ca2+ channel types with Ca2+-dependent afterhyperpolarizations in magnocellular supraoptic neurons.

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 Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ 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 Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 µM Cd2+ 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 Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ 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 Ca2+ 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 Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

Angiotensin AT1A receptors expressed in vasopressin-producing cells of the supraoptic nucleus contribute to osmotic control of vasopressin.

Angiotensin II (ANG) stimulates the release of arginine vasopressin (AVP) from the neurohypophysis through activation of the AT1 receptor within the brain, although it remains unclear whether AT1 receptors expressed on AVP-expressing neurons directly mediate this control. We explored the hypothesis that ANG acts through AT1A receptors expressed directly on AVP-producing cells to regulate AVP secretion. In situ hybridization and transgenic mice demonstrated localization of AVP and AT1A mRNA in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN), but coexpression of both AVP and AT1A mRNA was only observed in the SON. Mice harboring a conditional allele for the gene encoding the AT1A receptor (AT1Aflox) were then crossed with AVP-Cre mice to generate mice that lack AT1A in all cells that express the AVP gene (AT1AAVP-KO). AT1AAVP-KO mice exhibited spontaneously increased plasma and serum osmolality but no changes in fluid or salt-intake behaviors, hematocrit, or total body water. AT1AAVP-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 AT1A in AVP-producing cells of the SON but not the PVN, and a role for AT1A receptors in these cells in the osmotic regulation of AVP secretion.

Effects of lateral olfactory tract stimulation on Fos immunoreactivity in vasopressin neurones of the rat piriform cortex.

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.

Effect of dietary salt intake on epithelial Na+ channels (ENaC) in vasopressin magnocellular neurosecretory neurons in the rat supraoptic nucleus.

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, ß 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 ß- 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.

Spike patterning in oxytocin neurons: Capturing physiological behaviour with Hodgkin-Huxley and integrate-and-fire models.

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.

Osmotic activation of a Ca2+-dependent phospholipase C pathway that regulates ∆N TRPV1-mediated currents in rat supraoptic neurons.

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 Ca2+ that depends on influx through L-type Ca2+ channels. Our data therefore suggest that MNCs possess an osmotically-activated Ca2+-dependent PLC that contributes to the osmotic activation of ∆N TRPV1 and may therefore be important in MNC osmosensitivity and in central osmoregulation.

High-Sugar, but Not High-Fat, Food Activates Supraoptic Nucleus Neurons in the Male Rat.

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.