Hypothalamic Supraoptic Nuclei Research Articles

Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic control

BackgroundHypertension and obesity are highly interrelated diseases, being critical components of the metabolic syndrome. Despite the growing prevalence of this syndrome in the world population, efficient therapies are still missing. Thus, identification of novel targets and therapies are warranted. An enhanced activity of the hypothalamic renin-angiotensin system (RAS), including the recently discovered prorenin (PR) and its receptor (PRR), has been implicated as a common mechanism underlying aberrant sympatho-humoral activation that contributes to both metabolic and cardiovascular dysregulation in the metabolic syndrome. Still, the identification of precise neuronal targets, cellular mechanisms and signaling pathways underlying PR/PRR actions in cardiovascular- and metabolic related hypothalamic nuclei remain unknown. Methods and resultsUsing a multidisciplinary approach including patch-clamp electrophysiology, live calcium imaging and immunohistochemistry, we aimed to elucidate cellular mechanisms underlying PR/PRR actions within the hypothalamic supraoptic (SON) and paraventricular nucleus (PVN), key brain areas previously involved in cardiometabolic regulation. We show for the first time that PRR is expressed in magnocellular neurosecretory cells (MNCs), and to a lesser extent, in presympathetic PVN neurons (PVNPS). Moreover, we show that while PRR activation efficiently stimulates the firing activity of both MNCs and PVNPS neurons, these effects involved AngII-independent and AngII-dependent mechanisms, respectively. In both cases however, PR excitatory effects involved an increase in intracellular Ca2+ levels and a Ca2+-dependent inhibition of a voltage-gated K+ current. ConclusionsWe identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension.

Further evidence for a membrane receptor that binds glucocorticoids in the rodent hypothalamus

In parallel with their well-characterized delayed genomic effects, steroid hormones exhibit rapid, non-genomic effects at molecular, cellular and behavioral levels. We have proposed a model of rapid, non-genomic glucocorticoid inhibition of hypothalamic neuroendocrine cells through a putative membrane-associated glucocorticoid receptor (GR). Here we tested for plasma membrane GR immunoreactivity and binding in the hypothalamic supraoptic and paraventricular nuclei. Selective cross-linking of membrane proteins with membrane-impermeant BS3 and subsequent Western blot analysis with a monoclonal GR antibody revealed a reduction in the intensities of a ∼98kDa immunoreactive band and a ∼64kDa band in the rat paraventricular and supraoptic nuclei, and of a 64kDa band in hippocampal tissue, which suggested that these proteins are associated with the membrane. Saturation binding of [3H]-corticosterone and [3H]-dexamethasone in rat and mouse hypothalamic tissue revealed a Kd 4–24-fold lower and a Bmax 4–7-fold lower for the membrane-associated GR compared to the intracellular GR, suggesting a lower affinity and abundance of the glucocorticoid binding sites in the membrane than in the cytosol. Together, these findings suggest the presence of a low-affinity, low-abundance membrane-associated GR in the hypothalamus that shares homology with the intracellular GR, and are consistent with physiological evidence of rapid, non-genomic glucocorticoid actions in hypothalamic neuroendocrine cells that are GR dependent.

Reproductive Regulation of Gene Expression in the Hypothalamic Supraoptic and Paraventricular Nuclei.

Oxytocin secretion is required for successful reproduction. Oxytocin is synthesised by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei and the physiological demand for oxytocin synthesis and secretion is increased for birth and lactation. Therefore, we used a polymerase chain reaction (PCR) array screen to determine whether genes that might be important for synthesis and/or secretion of oxytocin are up- or down-regulated in the supraoptic and paraventricular nuclei of late-pregnant and lactating rats, compared to virgin rats. We then validated the genes that were most highly regulated using real time-quantitative PCR. Among the most highly regulated genes were those that encode for suppressors of cytokine signalling, which are intracellular inhibitors of prolactin signalling. Prolactin receptor activation changes gene expression via phosphorylation of signal transducer and activator of transcription 5 (STAT5). Using double-label immunohistochemistry, we found that phosphorylated STAT5 was expressed in almost all oxytocin neurones of late-pregnant and lactating rats but was almost absent from oxytocin neurones of virgin rats. We conclude that increased prolactin activation of oxytocin neurones might contribute to the changes in gene expression by oxytocin neurones required for normal birth and lactation.

Oxytocin in hypothalamic supraoptic nucleus is transferred to the caudate nucleus to influence pain modulation

Oxytocin (OXT), which is synthesized and secreted in the hypothalamic supraoptic nucleus (SON), is the most important bioactive substance in SON regulating pain process. Our previous study has pointed that OXT in the caudate nucleus (CdN) plays a role in pain modulation. The communication was designed to investigate the source of OXT in the rat CdN during pain process using the methods of push–pull perfusion and radioimmunoassay. The results showed that (1) pain stimulation increased the OXT concentration in the CdN perfusion liquid; (2) SON cauterization inhibited the increase of OXT concentration in CdN perfusion liquid induced by the pain stimulation, which role in both sides of SON cauterization was stronger than that in one side of SON cauterization; and (3) SON microinjection of l-glutamate sodium, which excited the SON neurons, increased OXT concentration in the CdN perfusion liquid. The data suggested that OXT in the CdN was influenced by SON during pain process, i.e., OXT in the SON might be transferred to the CdN to influence pain modulation.

Age-Related (Aged vs. Adult) Comparison of the Effect of Two Mild Stressors on the Nerve Growth Factor (NGF) in the Rat Hypothalamic Supraoptic Nucleus (SON) – Immunohistochemical Study

The ontogenetic period of life and stress can have different effects on the nerve growth factor (NGF) in the hypothalamus. The aim of our study was to investigate the influence of two mild stressors, acute and chronic exposure to forced swim (FS) or high-light open field (HL-OF), on neurons containing NGF. Immunofluorescence staining was used to reveal the density of NGF-immunoreactive (ir) cells in the hypothalamic supraoptic nucleus (SON) in adult (postnatal day 90; P90) and aged (P720) rats. The P90 and P720 rats that were subjected to acute and chronic FS showed no differences in the density of NGF-ir neurons in the SON compared with nonstressed rats. However, a significant increase in NGF-ir cells was noted after acute but not after chronic HL-OF only in P90 rats. What is more, there were no age-related (P90 vs. P720) changes in the density of NGF-ir neurons in non-stressed and FS- or HL-OF-stressed rats. Our results indicate that acute HL-OF was the only factor inducing changes in the density of NGF-ir neurons in the SON of adult rats. This could be related to the neuroprotective role of NGF-ir cells in response to acute HL-OF. The absence of age-dependent changes in the density of NGF-ir neurons may indicate that the ageing processes in SON do not generate changes in the NGF immunoreactivity of its neurons.

Recent Research on the Centrifugal Visual System in Mammalian Species

Ample evidence indicates that both retinofugal (classical visual and the retino-hypothalamic pathways) and retinopetal connections (centrifugal visual system) are found between the eye and the central nervous system. More than hundred years ago Ramon Y Cajal and Dogiel, whose names are very well known by neuro-anatomists, described the termination pattern of the fibers deriving from the avian central nervous system. However, the location of nerve cell bodies was not known at that time. In the last century many data accumulated about these neurons not only in lower vertebrates but in mammals as well. The structures where the neurons give rise to the centrifugal visual fibres in mammals are the following: reticular formation and raphe nuclei of the midbrain, superior colliculus, pretectum, gray matter of the midbrain, dentate gyrus, CA1 and CA3 regions of the hippocampus, olfactory tubercle, habenula, indusium griseum, hypothalamic supraoptic, Para ventricular and arcuate nuclei, and the lateral hypothalamus. The centrifugal visual fibers enter the optic nerve layer, then reach the inner plexiform layer and terminate in the inner nulear layer of the retina in the vicinity of the amacrine cells. A series of neuropeptides and neurotransmitters was described in the origin of the centrifugal visual system. These are the followings: luteinizing hormone releasing hormone, pituitary adenylate cyclase activating polypeptide, vasoactive intestinal polypeptide, serotonin, histamine and leu-enkephalin. Several hypotheses arose on the function of this system. Centrifugal visual system arising from the histaminergic mammillary neurons modifies the sleep/wake cycle. Hallucinogenic drugs through the limbic system may cause disturbance of visual function and result in seeing visual hallucinations or distorted images.

Vasopressin induces cholangiocyte proliferation in experimental cholestasis and in Polycystic Liver Disease

The hormone vasopressin (hereafter AVP) is a neuropeptide mainly synthesized in the brain’s hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, works by three distinct receptor subtypes: V1a, V1b, and V2 [1]. In liver, AVP is involved in glycogenolysis and neoglucogenesis and regenerative processes [2]. Cholangiocytes are the cells that line the biliary ducts and they are the target in a number of animal models of cholestasis including bile duct ligation (BDL) and in several human pathologies such as polycystic liver disease (PLD) characterized by the presence of numerous cysts within the liver that arise from biliary epithelium [3]. Since no data exist about the presence and the role of AVP and receptors in biliary epithelium, we aimed to evaluate the effects of AVP in experimental model of cholestasis and in course of PLD. In vivo, normal and BDL liver fragments from rats, normal and PLD from human patients were collected to evaluate: (i) intrahepatic bile duct mass (IBDM) by immunohistochemistry for citokeratin-19 (CK-19); and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from cystic epithelium) were stimulated with AVP in the absence/presence of antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT proliferation assay, cAMP levels by a RIA kit and the expression of some angiogenic factors, such as platelet-derived growth factor (PDGF) and Angiopoietins (Ang-1 and Ang-2). Cholangiocytes express V2 receptor that was upregulated following BDL and in course of polycystic disease. Treatment with AVP of cholangiocyte cultures increased proliferation, cAMP levels and expression of PDGF, Ang-1, Ang-2 in small cholangiocytes and LCDE cells. These increments were blocked by pre-incubation with the AVP antagonists. Our results showed that AVP play an important role in growth of the biliary epithelium during cholestasis and in cystic epithelium in course of PLD acting on the cAMP signalling pathway and increasing angiogenic factors. Additional studies are necessary, but these first results may be considered important in the regulation of the biliary growth/loss in course of cholangiopathies.

Patterns of Brain Activation and Meal Reduction Induced by Abdominal Surgery in Mice and Modulation by Rikkunshito.

Abdominal surgery inhibits food intake and induces c-Fos expression in the hypothalamic and medullary nuclei in rats. Rikkunshito (RKT), a Kampo medicine improves anorexia. We assessed the alterations in meal microstructure and c-Fos expression in brain nuclei induced by abdominal surgery and the modulation by RKT in mice. RKT or vehicle was gavaged daily for 1 week. On day 8 mice had no access to food for 6–7 h and were treated twice with RKT or vehicle. Abdominal surgery (laparotomy-cecum palpation) was performed 1–2 h before the dark phase. The food intake and meal structures were monitored using an automated monitoring system for mice. Brain sections were processed for c-Fos immunoreactivity (ir) 2-h after abdominal surgery. Abdominal surgery significantly reduced bouts, meal frequency, size and duration, and time spent on meals, and increased inter-meal interval and satiety ratio resulting in 92–86% suppression of food intake at 2–24 h post-surgery compared with control group (no surgery). RKT significantly increased bouts, meal duration and the cumulative 12-h food intake by 11%. Abdominal surgery increased c-Fos in the prelimbic, cingulate and insular cortexes, and autonomic nuclei, such as the bed nucleus of the stria terminalis, central amygdala, hypothalamic supraoptic (SON), paraventricular and arcuate nuclei, Edinger-Westphal nucleus (E-W), lateral periaqueduct gray (PAG), lateral parabrachial nucleus, locus coeruleus, ventrolateral medulla and nucleus tractus solitarius (NTS). RKT induced a small increase in c-Fos-ir neurons in the SON and E-W of control mice, and in mice with surgery there was an increase in the lateral PAG and a decrease in the NTS. These findings indicate that abdominal surgery inhibits food intake by increasing both satiation (meal duration) and satiety (meal interval) and activates brain circuits involved in pain, feeding behavior and stress that may underlie the alterations of meal pattern and food intake inhibition. RKT improves food consumption post-surgically that may involve modulation of pain pathway.

Distribution of oxytocin and co-localization with arginine vasopressin in the brain of mice.

Oxytocin (OT) and vasopressin (AVP) play a major role in social behaviours. Mice have become the species of choice for neurobiology of social behaviour due to identification of mouse pheromones and the advantage of genetically modified mice. However, neuroanatomical data on nonapeptidergic systems in mice are fragmentary, especially concerning the central distribution of OT. Therefore, we analyse the immunoreactivity for OT and its neurophysin in the brain of male and female mice (strain CD1). Further, we combine immunofluorescent detection of OT and AVP to locate cells co-expressing both peptides and their putative axonal processes. The results indicate that OT is present incellsof the neurosecretory paraventricular (Pa) and supraoptichypothalamicnuclei (SON). From the anterior SON, OTergic cells extend into the medial amygdala, where a sparse cell population occupies its ventral anterior and posterior divisions. Co-expression of OT and AVP in these nuclei is rare. Moreover, a remarkable OTergic cell group is found near the ventral bed nucleus of the stria terminalis (BST), distributed between the anterodorsal preoptic nucleus and the nucleus of anterior commissure (ADP/AC). This cell group, the rostral edge of the Pa and the periventricular hypothalamus display frequent OT+AVP double labelling, with a general dominance of OT over AVP immunoreactivity. Fibres with similar immunoreactivity profile innervate the accumbens shell and core, central amygdala and portions of the intervening BST. These data, together with data in the literature on rats, suggest that the projections of ADP/AC nonapeptidergic cells onto these brain centres could promote pup-motivated behaviours and inhibit pup avoidance during motherhood.

Distribution of vasotocin- and vasoactive intestinal peptide-like immunoreactivity in the brain of blue tit (Cyanistes coeruleus).

Blue tits (Cyanistes coeruleus) are songbirds, used as model animals in numerous studies covering a wide field of research. Nevertheless, the distribution of neuropeptides in the brain of this avian species remains largely unknown. Here we present some of the first results on distribution of Vasotocine (AVT) and Vasoactive intestinal peptide (VIP) in the brain of males and females of this songbird species, using immunohistochemistry mapping. The bulk of AVT-like cells are found in the hypothalamic supraoptic, paraventricular and suprachiasmatic nuclei, bed nucleus of the stria terminalis, and along the lateral forebrain bundle. Most AVT-like fibers course toward the median eminence, some reaching the arcopallium, and lateral septum. Further terminal fields occur in the dorsal thalamus, ventral tegmental area and pretectal area. Most VIP-like cells are in the lateral septal organ and arcuate nucleus. VIP-like fibers are distributed extensively in the hypothalamus, preoptic area, lateral septum, diagonal band of Broca. They are also found in the bed nucleus of the stria terminalis, amygdaloid nucleus of taenia, robust nucleus of the arcopallium, caudo-ventral hyperpallium, nucleus accumbens and the brainstem. Taken together, these results suggest that both AVT and VIP immunoreactive structures show similar distribution to other avian species, emphasizing evolutionary conservatism in the history of vertebrates. The current study may enable future investigation into the localization of AVT and VIP, in relation to behavioral and ecological traits in the brain of tit species.

Distinct vasopressin content in the hypothalamic supraoptic and paraventricular nucleus of rats exposed to low and high ambient temperature

Both high and low ambient temperature represent thermal stressors that, among other physiological responses, induce activation of the hypothalamic–pituitary–adrenal (HPA) axis and secretion of arginine-vasopressin (AVP). The exposure to heat also leads to disturbance of osmotic homeostasis. Since AVP, in addition to its well-known peripheral effects, has long been recognized as a hormone involved in the modulation of HPA axis activity, the aim of the present study was to elucidate the hypothalamic AVP amount in the acutely heat/cold exposed rats. Rats were exposed to high (+38°C) or low (+4°C) ambient temperature for 60min. Western blot was employed for determining hypothalamic AVP levels, and the difference in its content between supraoptic (SON) and paraventricular nucleus (PVN) was detected using immunohistochemical analysis. The results showed that exposure to both high and low ambient temperature increased hypothalamic AVP levels, although the increment was higher under heat conditions. On the other hand, patterns of AVP level changes in PVN and SON were stressor-specific, given that exposure to cold increased the AVP level in both nuclei, while heat exposure affected the PVN AVP content alone. In conclusion, our results revealed that cold and heat stress influence hypothalamic AVP amount with different intensity. Moreover, different pattern of AVP amount changes in the PVN and SON indicates a role of this hormone not only in response to heat as an osmotic/physical threat, but to the non-osmotic stressors as well.

The Profile of Exercise‐induced Neuronal Activation in Autonomic Control Nuclei Changes According to Thermoregulatory Phase

The purpose of this study was to examine whether the neuronal recruitment induced by exercise in autonomic nuclei is dependent of thermoregulatory phase and/or physical performance. Rats that were subjected to submaximal running exercise stopped in 3 different times: REST (control group), NADIR (the minimum heat dissipation phase), 20MIN (the stabilization phase of both body temperature ‐ Tb ‐ and tail skin temperature ‐ Tsk), and FATIGUE (Tb and Tsk reach their highest values​).The brains were removed and processed for determination of c‐Fos expression in the medial, median, and ventromedial preoptic areas (mPOA, mnPOA, and vmPOA, respectively), and paraventricular and supraoptic nucleus of hypothalamus (PVN and SON, respectively). Physical exercise induced an increase of c‐Fos expression in all brain areas. The c‐Fos expression in the vmPOA was similar for all exercising groups, while for mPOA, mnPOA, and PVN, it was gradually increased as the exercise and hyperthermic response are prolonged. Only after 20 min of exercise an increase in SON c‐Fos expression was verified. The neuronal recruitment within the mPOA, mnPOA, PVN, and SON was directly correlated to both physical performance and heat storage. The thermoregulatory brain areas are recruited even at small Tb changes, while the SON is recruited only when Tb reaches its higher values, possibly inducing changes on fluid balance. The recruitment rate in these areas is also influenced by duration of stress stimulus, either by increasing running time, as the prolonged hyperthermic response arising of exercise. Cnpq, Capes, and Fapemig.

Fructose Driven Vasopressin (VP) Secretion May Contribute to Chronic Kidney Diseases (CKD) under Chronic Dehydration

Chronic recurrent dehydration (WAN) can cause CKD, through a fructokinase (KHK) dependent way. VP is a peptide hormone that is synthesized in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), and is subsequently transported to and stored in posterior pituitary (PP), from where it is released into systemic circulation. VP may mediate some types of CKD through currently unclear mechanisms. Here we propose a novel mechanism for CKD resulted from chronic dehydration, that chronic dehydration may activate a local polyol pathway: aldose reductase (AR)‐fructose‐KHK, leading to elevated VP and thus worsening CKD. Current studies have shown existence of KHK mRNA in hypothalamic area and expression of KHK protein in both SON and PVN. The rise of copeptin, a serum marker of VP, was observed in wild type (WT)/WAN mice, but not in KHK‐KO/WAN mice. Further analysis revealed that VP storage in PP was elevated in WT/WAN mice compared with control WT mice, but was greatly reduced to about control level in KHK‐KO/WAN mice. KHK‐KO did not significantly affect VP storage in PP or serum copeptin levels in control mice. These results demonstrate that chronic dehydration may lead to CKD, at least partially, through elevated VP synthesis and secretion driven by activation of the AR‐fructose‐KHK polyol pathway. Future studies will further clarify the underlying mechanisms.

The median preoptic nucleus: front and centre for the regulation of body fluid, sodium, temperature, sleep and cardiovascular homeostasis.

Located in the midline anterior wall of the third cerebral ventricle (i.e. the lamina terminalis), the median preoptic nucleus (MnPO) receives a unique set of afferent neural inputs from fore-, mid- and hindbrain. These afferent connections enable it to receive neural signals related to several important aspects of homeostasis. Included in these afferent projections are (i) neural inputs from two adjacent circumventricular organs, the subfornical organ and organum vasculosum laminae terminalis, that respond to hypertonicity, circulating angiotensin II or other humoural factors, (ii) signals from cutaneous warm and cold receptors that are relayed to MnPO, respectively, via different subnuclei in the lateral parabrachial nucleus and (iii) input from the medulla associated with baroreceptor and vagal afferents. These afferent signals reach appropriate neurones within the MnPO that enable relevant neural outputs, both excitatory and inhibitory, to be activated or inhibited. The efferent neural pathways that proceed from the MnPO terminate on (i) neuroendocrine cells in the hypothalamic supraoptic and paraventricular nuclei to regulate vasopressin release, while polysynaptic pathways from MnPO to cortical sites may drive thirst and water intake, (ii) thermoregulatory pathways to the dorsomedial hypothalamic nucleus and medullary raphé to regulate shivering, brown adipose tissue and skin vasoconstriction, (iii) parvocellular neurones in the hypothalamic paraventricular nucleus that drive autonomic pathways influencing cardiovascular function. As well, (iv) other efferent pathways from the MnPO to sites in the ventrolateral pre-optic nucleus, perifornical region of the lateral hypothalamic area and midbrain influence sleep mechanisms.

Increased sociability and gene expression of oxytocin and its receptor in the brains of rats affected prenatally by valproic acid

Autism is a neurodevelopmental disorder characterised by the disruption of social interactions. Autistic animal models play a crucial role in neurophysiologic research on this disorder. One of these models is based on rats that have been prenatally treated with valproic acid — VPA rats. The aim of our study performed with this model was to investigate changes in sociability and gene expression of neuropeptides and receptors involved in regulating social behaviour. We focused on gene expression in the hypothalamus, where the neuropeptides oxytocin (OT) and arginine-vasopressin (AVP) are produced, as well as oxytocin receptors (OTR) in certain neuronal structures involved in the creation of social abilities. Our research showed that VPA rats spent more time in the part with an unknown animal and less time in the central part of a three chamber sociability test apparatus than control animals. The latency period of VPA rats before initiating social contact was decreased. In addition, during weaning, VPA female rats spent more time in direct interaction with an unknown rat. We also found that adult VPA rats had an increased expression of OT in the hypothalamic supraoptic and paraventricular nuclei and of OTR in the medial prefrontal cortex, piriform cortex, cortex–amygdala transition zone and the region of the basolateral and basomedial amygdaloid nuclei compared with controls.To sum up, we observed that a single prenatal injection of VPA increased social behaviour and gene expression of OT and OTR in neurological structures connected with the social behaviour of rats. One unanticipated finding was the absence of one of the core symptoms of autism in VPA rats, suggesting a decreased ability to understand intraspecific communication signals.

Carbon monoxide and nitric oxide interactions in magnocellular neurosecretory neurones during water deprivation.

Nitric oxide (NO) and carbon monoxide (CO) are diffusible gas messengers in the brain. Previously, we have shown their independent involvement in central fluid/electrolyte homeostasis control. In the present study, we investigated a possible functional interaction between NO/CO in the regulation of vasopressin (VP) and oxytocin (OT) magnocellular neurosecretory cells (MNCs) activity in euhydrated (EU) and dehydrated [48-h water-deprived (48WD)] rats. Using brain slices from EU and 48WD rats, we measured, by immunohistochemistry, the expression of neuronal NO synthase (nNOS, which synthesises NO) and haeme-oxygenase (HO-1, which synthesises CO) in the hypothalamic supraoptic nucleus (SON). In addition, we used patch-clamp electrophysiology to investigate whether regulation of SON MNC firing activity by endogenous CO was dependent on NO bioavailability and GABAergic inhibitory synaptic function. We found a proportion of OT and VP SON MNCs in EU rats to co-express both of HO-1 and nNOS (33.2±2.9% and 15.3±1.4%, respectively), which was increased in 48WD rats (55.5±0.9% and 21.0±1.7%, respectively, P<0.05 for both). Inhibition of endogenous HO activity [chromium mesoporphyrin IX chloride (CrMP) 20μm] induced MNC membrane hyperpolarisation and decreased firing activity, and these effects were blunted by previous blockade of endogenous NOS activity (l-NAME, 2mm) or blockade of inhibitory GABA function [Picrotoxin (Sigma-Aldrich, St Louis, MO, USA), 50μm]. No significant changes in SON NO bioavailability (4,5 diaminofluorescein diacetate fluorescence) were observed after CrMP treatment. Taken together, our results support a state-dependent functional inter-relationship between NO and CO in MNCs, in which CO acts as an excitatory gas molecule, whose effects are largely dependent on interactions with the inhibitory SON signals NO and GABA.

Regulation of Neuronal Activity in Hypothalamic Vasopressin Neurons.

Vasopressin is a peptide hormone secreted from the posterior pituitary gland in response to various physiological and/or pathological stimuli, including changes in body fluid volume and osmolality and stress exposure. Vasopressin secretion is controlled by the electrical activity of the vasopressinergic magnocellular neurosecretory cells located in the hypothalamic supraoptic nucleus and paraventricular nucleus. Vasopressin release can occur somatodendritically in the hypothalamus or at the level of pituitary axon terminals. The electrical activity of the vasopressin neurons assumes specific patterns of electrical discharge that are under the control of several factors, including the intrinsic properties of the neuronal membrane and synaptic and hormonal inputs. It is increasingly clear that glial cells perform critical signaling functions that contribute to signal transmission in neural circuits. Astrocytes contribute to neuronal signaling by regulating synaptic and extrasynaptic neurotransmission, as well as by mediating bidirectional neuronal-glial transmission. We recently discovered a novel form of neuronal-glial signaling that exploits the full spatial domain of astrocytes to transmit dendritic retrograde signals from vasopressin neurons to distal upstream neuronal targets. This retrograde trans-neuronal-glial transmission allows the vasopressin neurons to regulate their synaptic inputs by controlling upstream presynaptic neuron firing, thus providing a powerful means of controlling hormonal output.

Neuroendocrine regulation of lactation and milk production.

Prolactin (PRL) released from lactotrophs of the anterior pituitary gland in response to the suckling by the offspring is the major hormonal signal responsible for stimulation of milk synthesis in the mammary glands. PRL secretion is under chronic inhibition exerted by dopamine (DA), which is released from neurons of the arcuate nucleus of the hypothalamus into the hypophyseal portal vasculature. Suckling by the young activates ascending systems that decrease the release of DA from this system, resulting in enhanced responsiveness to one or more PRL-releasing hormones, such as thyrotropin-releasing hormone. The neuropeptide oxytocin (OT), synthesized in magnocellular neurons of the hypothalamic supraoptic, paraventricular, and several accessory nuclei, is responsible for contracting the myoepithelial cells of the mammary gland to produce milk ejection. Electrophysiological recordings demonstrate that shortly before each milk ejection, the entire neurosecretory OT population fires a synchronized burst of action potentials (the milk ejection burst), resulting in release of OT from nerve terminals in the neurohypophysis. Both of these neuroendocrine systems undergo alterations in late gestation that prepare them for the secretory demands of lactation, and that reduce their responsiveness to stimuli other than suckling, especially physical stressors. The demands of milk synthesis and release produce a condition of negative energy balance in the suckled mother, and, in laboratory rodents, are accompanied by a dramatic hyperphagia. The reduction in secretion of the adipocyte hormone, leptin, a hallmark of negative energy balance, may be an important endocrine signal to hypothalamic systems that integrate lactation-associated food intake with neuroendocrine systems.

Differences in acid-induced currents between oxytocin-mRFP1 and vasopressin-eGFP neurons isolated from the supraoptic and paraventricular nuclei of transgenic rats

The hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) consists of two types of magnocellular neurosecretory cells, oxytocin (OXT) and arginine vasopressin (AVP). We generated and characterized rats that express an OXT-monomeric red fluorescent protein 1 (mRFP1) and an AVP-enhanced green fluorescent protein (eGFP) fusion transgene. These transgenic rats enable the visualization of OXT or AVP neurons. Taking advantage of this, we examined the differences between OXT-mRFP1 neurons and AVP-eGFP neurons in response to acid. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive cationic channels that are activated by extracellular acidification. Although functional ASICs have been identified in AVP neurons, differences in acid-induced currents between OXT and AVP neurons in SON have not been reported. In the present study, we used the whole-cell patch-clamp technique to investigate differences between OXT-mRFP1 neurons and AVP-eGFP neurons reaction to acid in SON and PVN. In voltage clamp mode, lowering extracellular pH evoked inward currents in both OXT-mRFP1 neurons and AVP-eGFP neurons. In our findings, the acid-induced currents in the OXT-mRFP1 neurons were significantly smaller than those in the AVP-eGFP neurons. These acid-induced currents were inhibited by amiloride, a known blocker of ASICs. Further, to compare the response to acid between OXT-mRFP1 and AVP-eGFP neurons in the same transgenic rat, we used a double transgenic rat by mating an OXT-mRFP1 transgenic rat with an AVP-eGFP transgenic rat. The acid-induced currents of OXT-mRFP1 neurons were significantly smaller than those of AVP-eGFP neurons from the double transgenic rats. These currents were almost completely inhibited by amiloride. The difference of acid-sensitivity between OXT and AVP neurons might contribute to maintaining systematic order in hypothalamic function.

Short-term potentiation of GABAergic synaptic inputs to vasopressin and oxytocin neurones.

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.