Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin-synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin-synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin-synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin-immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin-immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1-deamino-8-d-arginine vasopressin via an osmotic mini-pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin-immunoreactive neurons, but not the number of oxytocin-immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin-synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

Alleviation of brain injury by applying TGN-020 in the supraoptic nucleus via inhibiting vasopressin neurons in rats of focal ischemic stroke.

AIMS: To understand mechanisms underlying vasopressin hypersecretion in stroke and its association with brain injury, we investigated effects of blocking aquaporin 4 (AQP4) in the supraoptic nucleus (SON) on vasopressin neuronal activity and cerebral injuries in male rats of unilateral middle cerebral artery occlusion (MCAO). MAIN METHODS: Establishing MCAO model without or with microinjection of TGN-020 into the SON, performing Western blots and immunohistochemistry and analyzing the expression levels and spatial distribution of functional proteins in the SON and/or the cerebral cortex. KEY FINDINGS: MCAO increased plasma vasopressin levels, caused neurological damage and increased glycogen synthase kinase 3ß (GSK-3ß) in the SON and the cortex of MCAO side. In the SON, MCAO significantly increased c-Fos in vasopressin neurons and astrocytic somata in the ventral glial lamina. MCAO significantly reduced glial fibrillary acidic protein (GFAP) and AQP4 around vasopressin neurons, which accompanied separation of GFAP from AQP4. By contrast, blocking AQP4 by microinjection of TGN-020 into the SON blocked MCAO-evoked GSK-3ß increase as well as the reduction of AQP4 relative to GFAP around vasopressin neurons in the SON. In the cortex, TGN-020 in the SON also blocked MCAO-evoked increase in GSK-3ß while reduced neurological damages. SIGNIFICANCE: These findings indicate that MCAO disrupts interactions of GFAP with AQP4 in astrocytic processes in the SON, which increases vasopressin neuronal activity. Blocking AQP4 in the SON can block abnormal activation of vasopressin neurons and alleviate ischemic brain injury, which provides novel targets for alleviating ischemic brain injury.

Alleviation of Cerebral Infarction of Rats With Middle Cerebral Artery Occlusion by Inhibition of Aquaporin 4 in the Supraoptic Nucleus.

In ischemic stroke, vasopressin hypersecretion is a critical factor of cerebral swelling and brain injury. To clarify neural mechanisms underlying ischemic stroke-evoked vasopressin hypersecretion, we observed the effect of unilateral permanent middle cerebral artery occlusion (MCAO) in rats on astrocytic plasticity and vasopressin neuronal activity in the supraoptic nucleus (SON) as well as their associated cerebral injuries. MCAO for 8 hr caused cerebral infarction in the MCAO side where water contents also increased. Immunohistochemical examination revealed that the percentage of phosphorylated extracellular signal-regulated protein kinase 1/2 (pERK1/2)-positive vasopressin neurons in the SON of MCAO side was significantly higher than that in non-MCAO side and in sham group. In the cortex, pERK1/2 and aquaporin 4 expressions increased significantly in the infarction area, while glial fibrillary acidic protein (GFAP) reduced significantly compared with the noninfarction side in brain cortex. Microinjection of N-(1,3,4-Thiadiazolyl)nicotinamide-020 [TGN-020, a specific blocker of aquaporin 4] into the SON blocked MCAO-evoked increases in pERK1/2 in the SON as well as the reduction of GFAP and the increase in pERK1/2 and aquaporin 4 in the infarction area of the cortex. Finally, oxygen and glucose deprivation reduced GFAP expression and the colocalization and molecular association of GFAP with aquaporin 4 in the SON in brain slices. These effects were blocked by TGN-020 and/or phloretin, a blocker of astrocytic volume-regulated anion channels. These findings indicate that blocking aquaporin 4 in the SON may reduce the activation of vasopressin neurons and brain injuries elicited by vasopressin during ischemic stroke.

Effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic supraoptic nucleus.

OBJECTIVE: The goal of this study was to reveal the impact of four types of atypical antipsychotics including amisulpride (AMI), olanzapine (OLA), quetiapine (QUE), and aripiprazole (ARI), with different receptor-affinity profile and dissociation constant, on the activity of hypothalamic supraoptic nucleus (SON) vasopressinergic and oxytocinergic neurons. METHODS: Male Sprague Dawley rats received a single injection of vehicle (VEH) (0.1 ml/100g), AMI (20 mg/kg), OLA (5 mg/kg), QUE (15 mg/kg/) or ARI (10 mg/kg). Ninety min after treatment, the animals were fixed by transcardial perfusion, the brains removed, and cryocut into serial coronal sections of 35 µm thickness. The sections were processed for c-Fos staining using an avidin-biotin-peroxidase complex and visualized by nickel intensified diaminobenzidine to reach black end product. Afterwards, the sections were exposed to vasopressin (AVP) and oxytocin (OXY) antibodies and the reaction product visualized by biotin-labeled fluorescent Alexa Fluor 568 dye. The data were evaluated from c-Fos and AVP or OXY merged sections. RESULTS: The present study shows that all four antipsychotics applied induced c-Fos expression in the SON. With respect to the stimulation efficacy of the individual antipsychotics, estimated based on the quantity of c-Fos-labeled AVP and OXY neurons, could be a preferential action assigned to QUE over moderate effect of ARI and lower effect to OLA and reduced effect of AMI (VEH < AMI < OLA < ARI < QUE). CONCLUSION: The present data for the first time provide an insight into the quantitative pattern of brain activity within the clusters of SON AVP and OXY cells in response to different atypical antipsychotics single treatment.

Peripheral insulin administration enhances the electrical activity of oxytocin and vasopressin neurones in vivo.

Oxytocin neurones are involved in the regulation of energy balance through diverse central and peripheral actions and, in rats, they are potently activated by gavage of sweet substances. Here, we test the hypothesis that this activation is mediated by the central actions of insulin. We show that, in urethane-anaesthetised rats, oxytocin cells in the supraoptic nucleus show prolonged activation after i.v. injections of insulin, and that this response is greater in fasted rats than in non-fasted rats. Vasopressin cells are also activated, although less consistently. We also show that this activation of oxytocin cells is independent of changes in plasma glucose concentration, and is completely blocked by central (i.c.v.) administration of an insulin receptor antagonist. Finally, we replicate the previously published finding that oxytocin cells are activated by gavage of sweetened condensed milk, and show that this response too is completely blocked by central administration of an insulin receptor antagonist. We conclude that the response of oxytocin cells to gavage of sweetened condensed milk is mediated by the central actions of insulin.

Oxytocin has sex-specific effects on social behaviour and hypothalamic oxytocin immunoreactive cells but not hippocampal neurogenesis in adult rats.

Oxytocin regulates social behaviours, pair bonding and hippocampal neurogenesis but most studies have used adult males. Our study investigated the effects of oxytocin on social investigation and adult hippocampal neurogenesis in male and female rats. Oxytocin has poor penetration of the blood-brain barrier, therefore we tested a nanoparticle drug, TRIOZAN™ (Ovensa Inc.), which permits greater blood-brain-barrier penetration. Adult male and female rats were injected daily (i.p.) for 10 days with either: oxytocin in PBS (0.5 or 1.0 mg/kg), oxytocin in TRIOZAN™ (0.5 or 1.0 mg/kg), or vehicle (PBS) and tested for social investigation. Oxytocin decreased body mass and increased social investigation and number of oxytocin-immunoreactive cells in the supraoptic nucleus (SON) of the hypothalamus in male rats only. In both sexes, oxytocin decreased the number of immature neurons (doublecortin+ cells) in the ventral hippocampus and reduced plasma 17ß-estradiol levels in a dose- and delivery-dependent way. Oxytocin in TRIOZAN™ reduced "sedation" observed post-injection and increased certain central effects (oxytocin levels in the hypothalamus and neurogenesis in the ventral hippocampus) relative to oxytocin in PBS, indicating that the nanoparticle may be used as an alternative brain delivery system. We showed that oxytocin has sex-specific effects on social investigation, body mass, "sedation", and the oxytocin system. In contrast, similar effects were observed in both sexes in neurogenesis and plasma 17ß-estradiol. Our work suggests that sex differences in oxytocin regulation of brain endpoints is region-specific (hypothalamus versus hippocampus) and that oxytocin does not promote social investigation in females.

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.

The spiking and secretory activity of oxytocin neurones in response to osmotic stimulation: a computational model.

KEY POINTS: A quantitative model of oxytocin neurones that combines a spiking model, a model of stimulus-secretion coupling and a model of plasma clearance of oxytocin was tested. To test the model, a variety of sources of published data were used that relate either the electrical activity of oxytocin cells or the secretion of oxytocin to experimentally induced changes in plasma osmotic pressure. To use these data to test the model, the experimental challenges involved were computationally simulated. The model predictions closely matched the reported outcomes of the different experiments. ABSTRACT: Magnocellular vasopressin and oxytocin neurones in the rat hypothalamus project to the posterior pituitary, where they secrete their products into the bloodstream. In rodents, both vasopressin and oxytocin magnocellular neurones are osmoresponsive, and their increased spiking activity is mainly a consequence of an increased synaptic input from osmoresponsive neurons in regions adjacent to the anterior wall of the third ventricle. Osmotically stimulated vasopressin secretion promotes antidiuresis while oxytocin secretion promotes natriuresis. In this work we tested a previously published computational model of the spiking and secretion activity of oxytocin cells against published evidence of changes in spiking activity and plasma oxytocin concentration in response to different osmotic challenges. We show that integrating this oxytocin model with a simple model of the osmoresponsive inputs to oxytocin cells achieves a strikingly close match to diverse sources of data. Comparing model predictions with published data using bicuculline to block inhibitory GABA inputs supports the conclusion that inhibitory inputs and excitatory inputs are co-activated by osmotic stimuli. Finally, we studied how the gain of osmotically stimulated oxytocin release changes in the presence of a hypovolaemic stimulus, showing that this is best explained by an inhibition of an osmotically regulated inhibitory drive to the magnocellular neurones.

Effects of salt-loading on supraoptic vasopressin neurones assessed by ClopHensorN chloride imaging.

Salt-loading (SL) impairs GABAA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K+ /Cl- co-transporter2 (KCC2) and up-regulating Na+ /K+ /Cl- co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl- ]i in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl- ]i in response to focal application of the GABAA agonist muscimol (100 µmol L-1 ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and ß-actin. Neurones that did not respond during chloride imaging did not show KCC2 and ß-actin protein expression. The KCC2 antagonist (VU0240551,10 µmol L-1 ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 µmol L-1 ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl- influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 µmol L-1 ) and protein kinase inhibitor (K252a) (100 nmol L-1 ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl- ]i in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.

A Common Neuroendocrine Substrate for Diverse General Anesthetics and Sleep.

How general anesthesia (GA) induces loss of consciousness remains unclear, and whether diverse anesthetic drugs and sleep share a common neural pathway is unknown. Previous studies have revealed that many GA drugs inhibit neural activity through targeting GABA receptors. Here, using Fos staining, ex vivo brain slice recording, and in vivo multi-channel electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of neuroendocrine cells, which are persistently and commonly activated by multiple classes of GA drugs. Remarkably, chemogenetic or brief optogenetic activations of these anesthesia-activated neurons (AANs) strongly promote slow-wave sleep and potentiates GA, whereas conditional ablation or inhibition of AANs led to diminished slow-wave oscillation, significant loss of sleep, and shortened durations of GA. These findings identify a common neural substrate underlying diverse GA drugs and natural sleep and reveal a crucial role of the neuroendocrine system in regulating global brain states. VIDEO ABSTRACT.

Estrogen receptor beta and G protein-coupled estrogen receptor 1 are involved in the acute estrogenic regulation of arginine-vasopressin immunoreactive levels in the supraoptic and paraventricular hypothalamic nuclei of female rats.

The ovarian hormone 17ß-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, ß and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor ß and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor ß and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats.

High salt loading increases brain derived neurotrophic factor in supraoptic vasopressin neurones.

High salt loading (SL) is associated with inappropriate arginine vasopressin (AVP) release and increased mean arterial pressure. Previous work has shown that chronic high salt intake impairs baroreceptor inhibition of rat AVP neurones through brain-derived neurotrophic factor (BDNF) dependent activation of tyrosine receptor kinase B (TrkB) and down-regulation of K+/Cl- co-transporter KCC2. This mechanism diminishes the GABAA inhibition of AVP neurones in the supraoptic nucleus (SON) by increasing intracellular chloride. However, the source of BDNF leading to this ionic plasticity is unknown. In the present study, we used adeno-associated viral vectors with short hairpin RNA against BDNF to test whether SON is the source of BDNF contributing to increased AVP release and elevated mean arterial pressure in high salt loaded rats. Virally mediated BDNF knockdown (shBDNF) in the SON of salt loaded rats significantly blocked the increases in BDNF mRNA and AVP heterogeneous RNA expression. The observed increase in the activation of TrkB receptor during salt loading is consistent with previous studies. Western blot analysis of SON punches revealed that tyrosine phosphorylation of TrkB (pTrkBY515) was significantly decreased in salt shBDNF rats compared to the salt scrambled (SCR) rats. Injections of shBDNF in the SON also significantly prevented the increase in plasma AVP concentration associated with salt loading. However, the salt loading induced increase in mean arterial pressure was not decreased with BDNF knockdown in the SON. Average daily fluid intake and urine output were significantly elevated in both salt SCR and salt shBDNF rats compared to the euhydrated controls. Daily average urine sodium concentration was significantly higher in shBDNF injected salt rats than the other groups. These findings indicate that BDNF produced in the SON contributes to the increased AVP secretion during high salt loading but not with respect to the subsequent increase in mean arterial pressure.

Effect of dietary salt intake on epithelial Na+ channels (ENaCs) in the hypothalamus of Dahl salt-sensitive rats.

All three epithelial Na+ channel (ENaC) subunits (α, ß, and γ) and the mineralocorticoid receptor (MR), a known regulator of ENaC, are located in vasopressin (VP) synthesizing magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. Our previous study showed that ENaC mediates a Na+ leak current that affects the steady-state membrane potential of VP neurons. This study was conducted in Dahl salt-sensitive (Dahl-SS) rats to determine if any abnormal responses in the expression of ENaC subunits and MR occur in the hypothalamus and kidney in response to a high dietary salt intake. After 21 days of high salt consumption, Dahl-SS rat resulted in a significant increase in γENaC expression and exhibited proteolytic cleavage of this subunit compared to Sprague-Dawley (SD) rats. Additionally, Dahl-SS rats had dense somato-dendritic γENaC immunoreactivity in VP neurons, which was absent in SD rats. In contrast, SD rats fed a high salt diet had significantly decreased αENaC subunit expression in the kidney and MR expression in the hypothalamus. Plasma osmolality measured daily for 22 days demonstrated that Dahl-SS rats fed a high salt diet had a steady increase in plasma osmolality, whereas SD rats had an initial increase that decreased to baseline levels. Findings from this study demonstrate that Dahl-SS rats lack a compensatory mechanism to down regulate ENaC during high dietary salt consumption, which may contribute to the development of hypertension.

Inhibition of lysine-specific demethylase enzyme disrupts sexually conditioned mate guarding in the female rat.

Although female rats are typically described as having a promiscuous mating strategy, if sexually naïve females have their formative sexually rewarding experiences paired with the same male, they will recognize that male and display mate-guarding behavior towards him in the presence of a female competitor. Female rats that display mate guarding behavior also show enhanced activation of oxytocin and vasopressin neurons in the supraoptic and paraventricular hypothalamic nucleus. Here, we examined the potential role that histone demethylation might have in establishing this pair-bonded behavior, and whether the corresponding changes in oxytocin and vasopressin neuronal activation depended on demethylation. To accomplish this, we examined the effect of a lysine-specific demethylase-1 inhibitor to block the action of demethylase enzymes and maintain the methylation state of corresponding genes. Female rats treated with the demethylase inhibitor failed to show any measure of mate guarding, whereas females treated with vehicle displayed mate guarding behavior. Demethylase inhibitor treatment also blocked the ability of familiar male cues to activate oxytocin and vasopressin neurons, whereas vehicle-treated females showed this enhanced activation. These data indicate that histone demethylation is a crucial component in the epigenetic modification of neural circuitry that underlies conditioned mate guarding in female rats. These results are the first to demonstrate the role of histone demethylation underlying changes in mating strategy.

Modulation of synaptic inputs in magnocellular neurones in a rat model of cancer cachexia.

In cancer cachexia, abnormal metabolism and neuroendocrine dysfunction cause anorexia, tissue damage and atrophy, which can in turn alter body fluid balance. Arginine vasopressin, which regulates fluid homeostasis, is secreted by magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus. Arginine vasopressin secretion by MNCs is regulated by both excitatory and inhibitory synaptic activity, alterations in plasma osmolarity and various peptides, including angiotensin II. In the present study, we used whole-cell patch-clamp recordings of brain slices to determine whether hyperosmotic stimulation and/or angiotensin II potentiate excitatory synaptic input in a rat model of cancer cachexia, similar to their effects in normal (control) rats. Hyperosmotic (15 and 60 mmol L-1   mannitol) stimulation and angiotensin II (0.1 µmol L-1 ) increased the frequency, but not the amplitude, of miniature excitatory postsynaptic currents in normal rats; in model rats, both effects were significantly attenuated. These results suggest that cancer cachexia alters supraoptic MNC sensitivity to osmotic and angiotensin II stimulation.

Dystrophin 71 and α1syntrophin in morpho-functional plasticity of rat supraoptic nuclei: Effect of saline surcharge and reversibly normal hydration.

Dystrophin (Dp) is a multidomain protein that links the actin cytoskeleton to the extracellular matrix through the dystrophin associated proteins complex (DAPC). Dp of 71 kDa (Dp71), corresponding to the COOH-terminal domain of dystrophin, and α1-syntrophin (α1Syn) as the principal component of the DAPC, are strongly expressed in the brain. To clarify their involvement in the central control of osmotic homeostasis, we investigated the effect of 14 days of salt loading (with drinking water containing 2% NaCl) and then reversibly to 30 days of normal hydration (with drinking water without salt), first on the expression by western-blotting and the distribution by immunochemistry of Dp71 and α1Syn in the SON of the rat and, second, on the level of some physiological parameters, as the plasma osmolality, natremia and hematocrit. Dp71 is the most abundant form of dystrophin revealed in the supraoptic nucleu (SON) of control rat. Dp71 was localized in magnocellular neurons (MCNs) and astrocytes, when α1Syn was observed essentially in astrocytes end feet. After 14 days of salt-loading, Dp71 and α1Syn signals decreased and a dual signal for these two proteins was revealed in the astrocytes processes SON surrounding blood capillaries. In addition, salt loading leads to an increase in plasma osmolality, natremia and hematocrit. Reversibly, after 30 days of normal hydration, the intensity of the signal for the two proteins, Dp71 and α1Syn, increased and approached that of control. Furtheremore, the levels of the physiological parameters decreased and approximated those of control. This suggests that Dp71 and α1Syn may be involved in the functional activity of the SON. Their localization in astrocyte end feet emphasizes their importance in neuronal-vascular-astrocyte interactions for the central detection of osmolality. In the SON, Dp71 and α1Syn may be involved in osmosensitivity.

Novel regulator of vasopressin secretion: phoenixin.

The newly described hypothalamic peptide, phoenixin, is produced in the hypothalamus and adenohypophysis, where it acts to control reproductive hormone secretion. Both phoenixin and its receptor GPR173 are expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, suggesting additional, nonreproductive effects of the peptide to control vasopressin (AVP) or oxytocin (OT) secretion. Hypothalamo-neurohypophysial explants released AVP but not OT in response to phoenixin. Intracerebroventricular administration of phoenixin into conscious, unrestrained male and female rats significantly increased circulating AVP, but not OT, levels in plasma, and it increased immediate early gene expression in the supraoptic nuclei of male rats. Bath application of phoenixin in hypothalamic slice preparations resulted in depolarization of PVN neurons, indicating a direct, neural action of phoenixin in the hypothalamus. Our results suggest that the newly described, hypothalamic peptide phoenixin, in addition to its effects on hypothalamic and pituitary mechanisms controlling reproduction, may contribute to the physiological mechanisms regulating fluid and electrolyte homeostasis.

Chronic Intranasal Oxytocin has Dose-dependent Effects on Central Oxytocin and Vasopressin Systems in Prairie Voles (Microtus ochrogaster).

Oxytocin (Oxt) is a neuropeptide with many functions, including modulation of social behavior(s) and anxiety. Due to its notable pro-social effects, it has been proposed as a treatment in the management of neuropsychiatric disorders, such as autism spectrum disorder (ASD), schizophrenia, and social anxiety; however, effects of long-term daily treatment are still being explored. Previously, we have shown that in male prairie voles (Microtus ochrogaster) exposure to Oxt during the peri-adolescent period impaired adult pair bonding in a dose-dependent fashion. In females, the medium dose used (0.8 IU/kg) appeared to facilitate pair bonding, and the low and medium doses were associated with fewer lines crossed in the open field. In this study, we examined central receptor binding and immunoreactive (IR) protein for Oxt and vasopressin (Avp), a closely related peptide. Voles were treated with saline vehicle, or one of three doses of Oxt (0.08, 0.8, 8.0 IU/kg) for three weeks from postnatal days 21 to 42, and euthanized as adults. We used autoradiography to examine Oxt and Avp receptor binding and immunohistochemistry to examine Oxt and Avp - IR cells in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Females that received the medium dose of Oxt had higher Oxt receptor binding in the nucleus accumbens shell (NAS), while males that received the medium dose had lower Avp-IR cells in the PVN. In summary, we found sex-specific effects of long-term exposure to intranasal Oxt on the Oxt and Avp systems at the weight-adjusted dose currently being used in clinical trials in humans.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

Possible involvement of central oxytocin in cisplatin-induced anorexia in rats.

During cancer chemotherapy, drugs such as 5-HT3 receptor antagonists have typically been used to control vomiting and anorexia. We examined the effects of oxytocin (OXT), which has been linked to appetite, on cisplatin-induced anorexia in rats. Fos-like immunoreactivity (Fos-LI) expressed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), the area postrema and the nucleus of the solitary tract (NTS) after intraperitoneal (ip) administration of cisplatin. We also examined the fluorescence intensity of OXT-mRFP1 after ip administration of cisplatin in OXT-mRFP1 transgenic rats. The mRFP1 fluorescence intensity was significantly increased in the SON, the PVN, and the NTS after administration of cisplatin. The cisplatin-induced anorexia was abolished by OXT receptor antagonist (OXTR-A) pretreatment. In the OXT-LI cells, cisplatin-induced Fos expression in the SON and the PVN was also suppressed by OXTR-A pretreatment. These results suggested that central OXT may be involved in cisplatin-induced anorexia in rats.