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.

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.

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.

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.

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.

Central angiotensin-(1-7) increases osmotic thirst.

NEW FINDINGS: What is the central question of this study? The central goal of this study was to understand the effects of central angiotensin-(1-7) on basal and osmotically stimulated water intake in rats. What is the main finding and its importance? This study demonstrated that central administration of angiotensin-(1-7) did not induce thirst in basal conditions but increased water intake after osmotic stimulation, such as water deprivation and salt loading. These results indicate a new function for this peptide, which, in turn, allows for future research on the mechanisms through which angiotensin-(1-7) influences osmotic thirst. Angiotensin-(1-7) [Ang-(1-7)] is generated by type 2 angiotensin-converting enzyme (ACE2) and binds to the MAS receptor. Although it is well known that Ang-(1-7) functionally antagonizes the effects of the classical renin-angiotensin system in several situations, the role of Ang-(1-7) in hydromineral homeostasis is not clear. The aim of this study was to assess the role of Ang-(1-7) on neuroendocrine responses to hyperosmolality in rats. Male Wistar rats were divided into the following three groups: control; 24 h of water deprivation (WD); and 24 h of salt loading (SL; 1.8% NaCl). Intracerebroventricular (i.c.v.) injections of Ang-(1-7) or vehicle were given to assess water intake and plasma concentration of vasopressin. Additionally, the brains from control and WD groups were collected to evaluate gene expression in the subfornical organ (SFO), paraventricular nucleus (PVN) and supraoptic nucleus (SON). It was found that i.c.v. Ang-(1-7) did not change water and salt intake in control rats; however, Ang-(1-7) increased water intake after WD and SL, with no change in salt intake. Plasma vasopressin was not changed by i.c.v. Ang-(1-7) in control or WD rats. Moreover, WD increased Mas gene expression in the SON and PVN, with no changes in Ace2 mRNA levels. In conclusion, Ang-(1-7) increases thirst after osmotic stimuli, indicating that a previous sensitization to its action is necessary. This finding is consistent with the increased Mas gene expression in the PVN and SON after water deprivation.

Perinatal exposure to organohalogen pollutants decreases vasopressin content and its mRNA expression in magnocellular neuroendocrine cells activated by osmotic stress in adult rats.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that produce neurotoxicity and neuroendocrine disruption. They affect the vasopressinergic system but their disruptive mechanisms are not well understood. Our group reported that rats perinatally exposed to Aroclor-1254 (A1254) and DE-71 (commercial mixtures of PCBs and PBDEs) decrease somatodendritic vasopressin (AVP) release while increasing plasma AVP responses to osmotic activation, potentially emptying AVP reserves required for body-water balance. The aim of this research was to evaluate the effects of perinatal exposure to A1254 or DE-71 (30mgkg/day) on AVP transcription and protein content in the paraventricular and supraoptic hypothalamic nuclei, of male and female rats, by in situ hybridization and immunohistochemistry. cFOS mRNA expression was evaluated in order to determine neuroendocrine cells activation due to osmotic stimulation. Animal groups were: vehicle (control); exposed to either A1254 or DE-71; both, control and exposed, subjected to osmotic challenge. The results confirmed a physiological increase in AVP-immunoreactivity (AVP-IR) and gene expression in response to osmotic challenge as reported elsewhere. In contrast, the exposed groups did not show this response to osmotic activation, they showed significant reduction in AVP-IR neurons, and AVP mRNA expression as compared to the hyperosmotic controls. cFOS mRNA expression increased in A1254 dehydrated groups, suggesting that the AVP-IR decrease was not due to a lack of the response to the osmotic activation. Therefore, A1254 may interfere with the activation of AVP mRNA transcript levels and protein, causing a central dysfunction of vasopressinergic system.

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.

Early oxytocin inhibition of salt intake after furosemide treatment in rats?

Body fluid homeostasis requires a complex suite of physiological and behavioral processes. Understanding of the role of the central nervous system (CNS) in integrating these processes has been advanced by research employing immunohistochemical techniques to assess responses to a variety of body fluid challenges. Such techniques have revealed sex/estrogen differences in CNS activation in response to hypotension and hypernatremia. In contrast, it has been difficult to conclusively identify specific CNS areas and neurotransmitter systems that are activated by hyponatremia using these techniques. In part, this difficulty is due to the temporal disconnect between the physiological effects of treatments commonly used to deplete body sodium and the behavioral response to such depletion. In some methods, sodium ingestion is delayed in association with increased oxytocin (OT), suggesting an inhibitory role for OT in sodium intake. Urinary sodium loss increases within an hour after treatment with furosemide, a natriuretic-diuretic, but sodium intake is delayed for 18-24h. Accordingly, we hypothesized that acute furosemide-induced sodium loss activates centrally-projecting OT neurons which provide an initial inhibition of sodium intake, and tested this hypothesis in ovariectomized Sprague-Dawley rats with or without estrogen using immunohistochemical methods. Neuronal activation in the hypothalamic paraventricular nuclei (PVN) after administration of furosemide corresponded to the timing of the physiological effects. The activation was not different in estrogen-treated rats, nor did estrogen alter the initial suppression of sodium intake. However, virtually no fos immunoreactive (fos-IR) neurons in the parvocellular PVN were also immunolabeled for OT. Thus, acute sodium loss after furosemide produces neural activation and an early inhibition of sodium intake that does not appear to involve activation of centrally-projecting OT neurons and is not influenced by estrogen.

NADPH-Diaphorase Colocalizes with GPER and Is Modulated by the GPER Agonist G1 in the Supraoptic and Paraventricular Nuclei of Ovariectomized Female Rats.

Nitric oxide is produced in the brain by the neuronal nitric oxide synthase (nNOS) and carries out a wide range of functions by acting as a neurotransmitter-like molecule. Gonadal hormones are involved in the regulation of the brain nitrergic system. We have previously demonstrated that estradiol, via classical estrogen receptors (ERs), regulates NOS activity in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus, acting through both ERα and ERß. Magnocellular and parvocellular neurons in the SON and PVN also express the G protein-coupled ER (GPER). In this study, we have assessed whether GPER is also involved in the regulation of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the SON and PVN. Adult female ovariectomized rats were treated with G1, a selective GPER agonist, or with G1 in combination with G15, a selective GPER antagonist. G1 treatment decreased NADPH-diaphorase expression in the SON and in all PVN subnuclei. The treatment with G1 + G15 effectively rescued the G1-dependent decrease in NADPH-diaphorase expression in both brain regions. In addition, the activation of extracellular signal-regulated kinase (ERK) 1/2, one of the kinases involved in the GPER-dependent intracellular signaling pathway and in NOS phosphorylation, was assessed in the same brain nuclei. Treatment with G1 significantly decreased the number of p-ERK 1/2-positive cells in the SON and PVN, while the treatment with G1 + G15 significantly recovered its number to control values. These findings suggest that the activation of GPER in the SON and PVN inhibits the phosphorylation of ERK 1/2, which induces a decrease in NADPH-diaphorase expression.

The effects of long-term estradiol treatment on social behavior and gene expression in adult female rats.

This study tested the effects of long-term estradiol (E2) replacement on social behavior and gene expression in brain nuclei involved in the regulation of these social behaviors in adult female rats. We developed an ultrasonic vocalization (USV) test and a sociability test to examine communications, social interactions, and social preference, using young adult female cagemates. All rats were ovariectomized (OVX) and implanted with a Silastic capsule containing E2 or vehicle, and housed in same-treatment pairs for a 3-month period. Then, rats were behaviorally tested, euthanized, and 5 nuclei in the brain's social decision-making circuit were selected for neuromolecular profiling by a multiplex qPCR method. Our novel USV test proved to be a robust tool to measure numbers and types of calls emitted by cagemates that had been reintroduced after a 1-week separation. Results also showed that E2-treated OVX rats had profoundly decreased numbers of USV calls compared to vehicle-treated OVX rats. In a test of sociability, in which a female was allowed to choose between her cagemate or a same-treatment novel rat, we found few effects of E2 compared to vehicle, although interestingly, rats chose the cagemate over an unfamiliar conspecific. Gene expression results revealed that the supraoptic nucleus had the greatest number of gene changes caused by E2: Oxt, Oxtr and Avp were increased, and Drd2, Htr1a, Grin2b, and Gabbr1 were decreased, by E2. No genes were affected in the prefrontal cortex, and 1-4 genes were changed in paraventricular nucleus (Pgr), bed nucleus of the stria terminalis (Oxtr, Esr2, Dnmt3a), and medial amygdala (Oxtr, Ar, Foxp1, Tac3). Thus, E2 changes communicative interactions between adult female rats, together with selected expression of genes in the brain, especially in the supraoptic nucleus.

Oestradiol effects on neuroendocrine responses induced by water deprivation in rats.

Water deprivation (WD) induces changes in plasma volume and osmolality, which in turn activate several responses, including thirst, the activation of the renin-angiotensin system (RAS) and vasopressin (AVP) and oxytocin (OT) secretion. These systems seem to be influenced by oestradiol, as evidenced by the expression of its receptor in brain areas that control fluid balance. Thus, we investigated the effects of oestradiol treatment on behavioural and neuroendocrine changes of ovariectomized rats in response to WD. We observed that in response to WD, oestradiol treatment attenuated water intake, plasma osmolality and haematocrit but did not change urinary volume or osmolality. Moreover, oestradiol potentiated WD-induced AVP secretion, but did not alter the plasma OT or angiotensin II (Ang II) concentrations. Immunohistochemical data showed that oestradiol potentiated vasopressinergic neuronal activation in the lateral magnocellular PVN (PaLM) and supraoptic (SON) nuclei but did not induce further changes in Fos expression in the median preoptic nucleus (MnPO) or subfornical organ (SFO) or in oxytocinergic neuronal activation in the SON and PVN of WD rats. Regarding mRNA expression, oestradiol increased OT mRNA expression in the SON and PVN under basal conditions and after WD, but did not induce additional changes in the mRNA expression for AVP in the SON or PVN. It also did not affect the mRNA expression of RAS components in the PVN. In conclusion, our results show that oestradiol acts mainly on the vasopressinergic system in response to WD, potentiating vasopressinergic neuronal activation and AVP secretion without altering AVP mRNA expression.

Estradiol and angiotensin II crosstalk in hydromineral balance: Role of the ERK1/2 and JNK signaling pathways.

The angiotensin II (ANGII) receptor AT1 plays an important role in the control of hydromineral balance, mediating the dipsogenic and natriorexigenic effects and neuroendocrine responses of ANGII. While estradiol (E2) is known to modulate several actions of ANGII in the brain, the molecular and cellular mechanisms of the interaction between E2 and ANGII and its physiological role in the control of body fluids remain unclear. We investigated the influence of E2 (40 µg/kg) pretreatment and extracellular-signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) cell signaling on the dipsogenic and natriorexigenic effects, as well as the neuroendocrine responses to angiotensinergic central stimulation in ovariectomized rats (OVX). We showed that the inhibitory effect of E2 on ANGII-induced water and sodium intake requires the ERK1/2 and JNK signaling pathways. On the other hand, E2 pretreatment prevents the ANGII-induced phosphorylation of ERK and JNK in the lamina terminalis. E2 therapy decreased oxytocin (OT) and vasopressin (AVP) secretion and decreased ERK1/2 phosphorylation in the supraoptic and paraventricular nuclei (SON and PVN, respectively). We found that the AVP secretion induced by ANGII required ERK1/2 signaling, but OT secretion did not involve ERK1/2 signaling. Taken together, these results demonstrate that E2 modulates ANGII-induced water and sodium intake and AVP secretion by affecting the ERK1/2 and JNK pathways in the lamina terminalis and ERK1/2 signaling in the hypothalamic nuclei (PVN and SON) in OVX rats.

Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation.

BACKGROUND: Rasd1 is a member of the Ras family of monomeric G proteins that was first identified as a dexamethasone inducible gene in the pituitary corticotroph cell line AtT20. Using microarrays we previously identified increased Rasd1 mRNA expression in the rat supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in response to increased plasma osmolality provoked by fluid deprivation and salt loading. RASD1 has been shown to inhibit adenylyl cyclase activity in vitro resulting in the inhibition of the cAMP-PKA-CREB signaling pathway. Therefore, we tested the hypothesis that RASD1 may inhibit cAMP stimulated gene expression in the brain. RESULTS: We show that Rasd1 is expressed in vasopressin neurons of the PVN and SON, within which mRNA levels are induced by hyperosmotic cues. Dexamethasone treatment of AtT20 cells decreased forskolin stimulation of c-Fos, Nr4a1 and phosphorylated CREB expression, effects that were mimicked by overexpression of Rasd1, and inhibited by knockdown of Rasd1. These effects were dependent upon isoprenylation, as both farnesyltransferase inhibitor FTI-277 and CAAX box deletion prevented Rasd1 inhibition of cAMP-induced gene expression. Injection of lentiviral vector into rat SON expressing Rasd1 diminished, whereas CAAX mutant increased, cAMP inducible genes in response to osmotic stress. CONCLUSIONS: We have identified two mechanisms of Rasd1 induction in the hypothalamus, one by elevated glucocorticoids in response to stress, and one in response to increased plasma osmolality resulting from osmotic stress. We propose that the abundance of RASD1 in vasopressin expressing neurons, based on its inhibitory actions on CREB phosphorylation, is an important mechanism for controlling the transcriptional responses to stressors in both the PVN and SON. These effects likely occur through modulation of cAMP-PKA-CREB signaling pathway in the brain.

Testing the critical window of estradiol replacement on gene expression of vasopressin, oxytocin, and their receptors, in the hypothalamus of aging female rats.

The current study tested the "critical window" hypothesis of menopause that postulates that the timing and duration of hormone treatment determine their potential outcomes. Our focus was genes in the rat hypothalamus involved in social and affiliative behaviors that change with aging and/or estradiol (E2): Avp, Avpr1a, Oxt, Oxtr, and Esr2 in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Rats were reproductively mature or aging adults, ovariectomized, given E2 or vehicle treatment of different durations, with or without a post-ovariectomy delay. Our hypothesis was that age-related changes in gene expression are mitigated by E2 treatments. Contrary to this, PVN Oxtr increased with E2, and Avpr1a increased with age. In the SON, Avpr1a increased with age, Oxtr with age and timing, and Avp was altered by duration. Thus, chronological age and E2 have independent actions on gene expression, with the "critical window" hypothesis supported by the observed timing and duration effects.

Presynaptic G Protein-Coupled Receptors Differentially Modulate Spontaneous Glutamate Release in the Supraoptic Nucleus.

Spontaneous glutamate release in the supraoptic nucleus is modulated by a number of inhibitory G protein coupled receptors (GPCR), including GABAB , adenosine A1 and group III metabotropic glutamate receptors (mGluR). It remains unclear whether they have distinct roles or are redundant mechanisms that protect from hyperexcitation. To address this question, we facilitated spontaneous glutamate release using nifedipine or forskolin, which act in a protein kinase A (PKA)-independent and -dependent manner, respectively, and tested the effects of inhibitory GPCR agonists. We found that a GABAB receptor (GABAB R) agonist specifically inhibited forskolin-induced miniature excitatory postsynaptic currents (mEPSC), in contrast to an adenosine A1 receptor (A1R) agonist, which specifically inhibited nifedipine-induced mEPSCs. This suggests that GABAB Rs and A1 Rs modulate independent mechanisms activated by forskolin and nifedipine, respectively. However, the inhibitory effects of GABAB R and A1 R agonists on basal mEPSCs occluded each other, suggesting that these receptors also have an overlapping role. Group III mGluRs appear to have a greater control over glutamate release because agonists to these receptors inhibited both nifedipine- and forskolin-induced mEPSCs. mEPSCs induced by norepinephrine had the same characteristics as those induced by forskolin [i.e. PKA-dependence and sensitivity to GABAB R and group III mGluR agonists, but not an A1 R agonist]. In summary, the present study highlights the differential effects of GABAB R, A1 R and mGluR agonists on glutamate release stimulated by different secretagogues, including the endogenous neuromodulator norepinephrine. These results suggest that the roles of these inhibitory GPCRs are not completely redundant, and also indicate the physiological implications of having different excitatory and inhibitory GPCRs on the same synapse.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.

BACKGROUND: Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression. RESULTS: The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress. CONCLUSION: Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.