Vasopressin Neurons Research Articles

SAT-427 Low Chloride Transporter Expression in Vasopressin Neurons

Several membrane proteins function as ion pumps/transporters that work to maintain the relative ionic concentrations necessary for neuronal signaling. The potassium-chloride co-transporter two (KCC2) is a symporter that actively pumps K+ and Cl- out of the cell in order to maintain a low intracellular [Cl-], while the sodium-potassium-chloride cotransporters one and two (NKCC1/NKCC2) are symporters that actively pump Na+, K+, and Cl- into the cell. KCC2 and NKCC1/2, therefore, have opposing actions on Cl- transport. In the adult, high KCC2 expression leads to low intracellular [Cl-], which causes GABAA receptor Cl- channels to flux Cl- into the neuron, causing a membrane hyperpolarization. Early in development however, low KCC2 expression reverses the Cl- gradient and causes GABAA receptors to flux Cl- out of the neuron, leading to membrane depolarization and neuronal excitation. The change in the polarity of GABA signaling during development is, in part, due to a shift in KCC2 expression from low to high with the maturation of synaptic circuits. Our previous studies have demonstrated an excitatory GABAA receptor-mediated response due to a GABA equilibrium potential (EGABA) that is shifted positive, like that seen early in development, in adult vasopressin (VP)-secreting neurons of the rat hypothalamus. In the current study, we used immunohistochemistry to compare expression levels of the main Cl- transporters, KCC2, NKCC1, and NKCC2, between VP- and oxytocin (OT)-secreting neurons of the hypothalamic paraventricular and supraoptic nuclei. We found that, in adult male Wistar rats, expression of all three transporters is uniformly lower in VP neurons than in adjacent OT neurons in the hypothalamus. Because the phosphorylation state of KCC2 affects membrane trafficking and stabilization, we are also using Western blot to analyze phosphorylation of KCC2 under different osmotic conditions. Finally, we are using Cre-dependent viral transduction to express a light-activatable Cl- channel conditionally in vasopressin-secreting neurons to test for the effect of Cl- flux on vasopressin secretion.

SUN-473 The Contribution of Bmal1 Expression in the Suprachiasmatic Nucleus to Female Fertility

The preovulatory gonadotropin-releasing hormone (GnRH) and resulting luteinizing hormone (LH) surge is gated to the onset of the active period in mice by a circadian and steroid signal. The temporal signal that initiates this process is believed to arrive from the suprachiasmatic nucleus (SCN), the hypothalamic circadian pacemaker. The SCN may contribute to reproduction through two discrete projections: vasoactive inhibitory peptide (VIP) neurons in the SCN project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and arginine vasopressin (AVP) neurons in the SCN project to kisspeptin (Kiss1) neurons in the anteroventral periventricular (AVPV). Genomic deletion of the critical circadian clock component, Bmal1, results in infertility in females. We sought to determine whether Bmal1 was critical in the SCN VIP and AVP neurons by using cre/lox technology to create three lines of mutant mice: AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/-. We found no significant difference in LH levels following a kisspeptin challenge (2 mg/kg) between AVP-Bmal1-/- and Bmal1flox/flox (617.4 ± 216.9 vs 629.6 ± 110.6 pg/mL) or GnRH challenge (10 μg/kg) (1029.0 ± 246.2 vs 712.7 ± 142.6 pg/mL). Preliminary findings of an induced LH surge found AVP-Bmal1-/- and VIP-Bmal1-/- mice are able to mount a surge at the time of lights off (Bmal1flox/flox: 3 of 5; AVP-Bmal1-/-: 1 of 2, VIP-Bmal1:-/- 4 of 4; AVP-VIP-Bmal1-/- 0 of 2). Additionally, we were able to detect an endogenous surge at the time of lights off in both AVP-Bmal1-/-, VIP-Bmal1-/- mice, but not AVP-VIP-Bmal1-/-. However, AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/- females carried litters to term and show no differences in the time to first litter compared to Bmal1flox/flox control (AVP-Bmal1-/-: 48 ± 4.8 vs 44.3 ± 0.3 days; VIP-Bmal1-/-: 29.6 ± 7.7 vs 31.0 ± 4.2 days; AVP-VIP-Bmal1-/-;27.7 ± 7 vs 23 ± 0 days); number of litters in 100 days (AVP-Bmal1-/-: 3.7 ±0.6 vs 4 ± 0.6; VIP-Bmal1-/-: 3.0 ± 1.0 vs 3.0 ± 0.0; AVP-VIP-Bmal1-/- 3.5 ± 0.7 vs 3.5 ± 0.7 days); or pups per litter (AVP-Bmal1-/-: 7.2 ± 0.7 vs 7.3 ± 0.7; VIP-Bmal1-/-: 6.2 ± 1.6 vs 7.6 ± 2.9; AVP-VIP-Bmal1-/- 8.1 ± 0.9 vs 9 ± 1.4 pups). Overall, these findings suggest that Bmal1 expression in the SCN AVP or VIP neurons is not required for fertility in a 12:12 light:dark cycle.

OR06-4 Brain Derived Neurotrophic Factor in the Regulation of Supraoptic Vasopressin Neurons in Development of Hyponatremia

Dilutional hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in patients with liver failure. The current conventional therapies are palliative and frequently ineffective. The neural mechanisms causing dysregulation of AVP secretion are not completely understood. Based on previous studies, we hypothesis that inappropriate AVP release associated with liver failure is due to increased Brain Derived Neurotrophic Factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABAA inhibition in SON AVP neurons by increasing intracellular chloride ([Cl]i) through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl- co-transporter 2 (KCC2). This loss of inhibition could increase AVP secretion. To test this hypothesis, chronic bile duct ligated (BDL) rats, a model of dilutional hyponatremia associated with liver failure, were injected with shRNA to prevent increased BDNF in the SON. Adult male Sprague Dawley rats were bilaterally injected in the SON with AAV2- shRNA-BDNF-mCherry or AAV2- shRNA-SCR-mCherry. After two weeks, the rats had BDL or sham ligation surgery. Four weeks later, SON regions were collected by punches for western blot analysis. Fluid accumulation, liver and body weight were recorded to verify if the rats develop liver failure. Blood samples were collected to measure plasma osmolality, hematocrit, and circulating AVP concentration. Data were analyzed by one-way ANOVA with Bonferroni comparisons. The BDL rats had significant increases in liver to body weight ratio (p< 0.05; 6-9) compared to sham rats indicating liver failure and ascites with bile duct ligation. BDL rats with scrambled injections (BDL SCR) developed hyponatremia with significant decrease in plasma osmolality and hematocrit along with significant increase in plasma AVP concentration (all p< 0.05; 6-9) compared to sham groups. This is the first study to show that phosphorylation of TrkB (pTrkBY515) is significantly increased along with significant decrease in phosphorylation of KCC2 (pKCC2S940) in BDL SCR rats compared to the sham rats (p< 0.05; 6-8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) prevented the rats from developing hyponatremia and caused significant increase in plasma osmolality and hematocrit compared to BDL SCR rats (p< 0.05; 6-9). The BDL shBDNF rats had significant (p< 0.05; 6-9) decreases in plasma AVP concentration compared to BDL SCR rats. shBDNF injections in the SON of BDL rats significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p< 0.05;6-8) associated with bile duct ligation. mCherry expression was used to verify the specificity of stereotaxic injections. Rats that did not had successful virus injections in the SON were analyzed separately. The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver cirrhosis.

Intracellular Chloride Regulation of Supraoptic Vasopressin Neurons during Salt Loading

Salt loading (SL) increases intracellular chloride concentration [Cl]i, impairing GABAA inhibition of arginine vasopressin (AVP) neurons in the supraoptic nucleus (SON) of hypothalamus. But the regulatory mechanisms leading to increased [Cl]i is not completely understood. Based on previous studies, we hypothesize that SL activates tyrosine receptor kinase B (TrkB) and downregulates K+/Cl− co‐transporter 2 (KCC2) membrane expression. Downregulation of KCC2 decreases the efflux of chloride ion causing increase in [Cl]i in SON AVP neurons. In this study, we have combined virally mediated ClopHensorN, a relatively new ratiometric chloride imaging technique with capillary based Simple Western technology to record changes in [Cl]i and specifically detect KCC2 protein expression in individual SON AVP neurons.Adult male Sprague Dawley rats were bilaterally injected in the SON with rAAV2‐0VP1‐ClopHensorN. The ClopHensorN (Addgene Plasmid #50758 from Colin Akerman) was packaged in an AAV2 vector with a AVP specific promotor (Addgene Plasmid #40868 from Harold Gainer). After 2 weeks, the rats were given either water or 2% NaCl to drink for 7 days. At the end of the protocol, the rats were anesthetized with inactin and their SONs were dissociated. The cells were plated on coverslips and placed in a perfusion bath on an inverted microscope for ratiometric live cell imaging. ClopHensorN positive neurons were tested for decrease or increase in [Cl]i to focal application of GABAA agonist muscimol (100uM). After imaging, individual neurons were collected by aspirating into a patch pipette to verify KCC2 and ß‐Actin protein expression. The standard matrix (12–230kDa) in Protein Simple Wes was used to identify and quantitate very low concentration of protein from single neuron. Data were analyzed by Chi‐squared test and one‐way ANOVA with Bonferroni comparisons.Muscimol application to SL SONs either significantly increased chloride efflux (p&lt;0.05;13/20) or did not change chloride flux (7/20). TrkB antagonist (AnA,50uM) significantly blocked the chloride effluxes. SONs from euhydrated rats showed muscimol induced chloride influx (p&lt;0.05;11/16). KCC2 antagonist (VU 0240551,10uM) significantly blocked the chloride influxes in euhydrated rats. SON AVP neurons that responded to pharmacological inhibitors during chloride imaging were viable and expressed KCC2 co‐transporter and ß‐Actin. Neurons that did not respond during chloride imaging did not had KCC2 and ß‐Actin protein expression. Salt loading increases [Cl]i in SON AVP neurons through TrKB‐KCC2 dependent mechanism.Support or Funding InformationSupported by R01 HL119458 and 18PRE34060035This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

Electrophysiological properties of identified oxytocin and vasopressin neurones.

To understand the contribution of intrinsic membrane properties to the different in vivo firing patterns of oxytocin (OT) and vasopressin (VP) neurones, in vitro studies are needed, where stable intracellular recordings can be made. Combining immunochemistry for OT and VP and intracellular dye injections allows characterisation of identified OT and VP neurones, and several differences between the two cell types have emerged. These include a greater transient K+ current that delays spiking to stimulus onset, and a higher Na+ current density leading to greater spike amplitude and a more stable spike threshold, in VP neurones. VP neurones also show a greater incidence of both fast and slow Ca2+ -dependent depolarising afterpotentials, the latter of which summate to plateau potentials and contribute to phasic bursting. By contrast, OT neurones exhibit a sustained outwardly rectifying potential (SOR), as well as a consequent depolarising rebound potential, not found in VP neurones. The SOR makes OT neurones more susceptible to spontaneous inhibitory synaptic inputs and correlates with a longer period of spike frequency adaptation in these neurones. Although both types exhibit prominent Ca2+ -dependent afterhyperpolarising potentials (AHPs) that limit firing rate and contribute to bursting patterns, Ca2+ -dependent AHPs in OT neurones selectively show significant increases during pregnancy and lactation. In OT neurones, but not VP neurones, AHPs are highly dependent on the constitutive presence of the second messenger, phosphatidylinositol 4,5-bisphosphate, which permissively gates N-type channels that contribute the Ca2+ during spike trains that activates the AHP. By contrast to the intrinsic properties supporting phasic bursting in VP neurones, the synchronous bursting of OT neurones has only been demonstrated in vitro in cultured hypothalamic explants and is completely dependent on synaptic transmission. Additional differences in Ca2+ channel expression between the two neurosecretory terminal types suggests these channels are also critical players in the differential release of OT and VP during repetitive spiking, in addition to their importance to the potentials controlling firing patterns.

Endoplasmic reticulum stress induces apoptosis of arginine vasopressin neurons in central diabetes insipidus via PI3K/Akt pathway.

AimsCentral diabetes insipidus (CDI), a typical complication caused by pituitary stalk injury, often occurs after surgery, trauma, or tumor compression around hypothalamic structures such as the pituitary stalk and optic chiasma. CDI is linked to decreased arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus, along with a deficit in circulating AVP and oxytocin. However, little has been elucidated about the changes in AVP neurons in CDI. Hence, our study was designed to understand the role of several pathophysiologic changes such as endoplasmic reticulum (ER) stress and apoptosis of AVP neurons in CDI.MethodsIn a novel pituitary stalk electric lesion (PEL) model to mimic CDI, immunofluorescence and immunoblotting were used to understand the underlying regulatory mechanisms.ResultsWe reported that in CDI condition, generated by PEL, ER stress induced apoptosis of AVP neurons via activation of the PI3K/Akt and ERK pathways. Furthermore, application of N‐acetylcysteine protected hypothalamic AVP neurons from ER stress‐induced apoptosis through blocking the PI3K/Akt and ERK pathways.ConclusionOur findings showed that AVP neurons underwent apoptosis induced by ER stress, and ER stress might play a vital role in CDI condition through the PI3K/Akt and ERK pathways.

Role of Connexin 36 in Autoregulation of Oxytocin Neuronal Activity in Rat Supraoptic Nucleus.

In the supraoptic nucleus (SON), the incidence of dye coupling among oxytocin (OT) neurons increases significantly in nursing mothers. However, the type(s) of connexin (Cx) involved is(are) unknown. In this study, we specifically investigated whether Cx36 plays a functional role in the coupling between OT neurons in the SON of lactating rats. In this brain region, Cx36 was mainly coimmunostained with vasopressin neurons in virgin female rats, whereas in lactating rats, Cx36 was primarily colocalized with OT neurons. In brain slices from lactating rats, application of quinine (0.1 mM), a selective blocker of Cx36, significantly reduced dye coupling among OT neurons as well as the discharge/firing frequency of spikes/action potentials and their amplitude, and transiently depolarized the membrane potential of OT neurons in whole-cell patch-clamp recordings. However, quinine significantly reduced the amplitude, but not frequency, of inhibitory postsynaptic currents in OT neurons; the duration of excitatory postsynaptic currents was reduced but not their frequency and amplitude. Furthermore, the excitatory effect of OT (1 pM) on OT neurons was significantly weakened and delayed by quinine, and burst firing was absent in the presence of this inhibitor. Lastly, Western blotting analysis revealed that the presence of combined, but not alone, quinine and OT significantly reduced the amount of Cx36 in the SON. Thus, Cx36-mediated junctional communication plays a crucial role in autoregulatory control of OT neuronal activity, likely by acting at the postsynaptic sites. The level of Cx36 is modulated by its own activity and the presence of OT.

Osmotic stress induces corticosteroid-binding globulin expression in the rat hypothalamo-hypophyseal system

Corticosteroid-binding globulin CBG is expressed in magnocellular hypothalamic nuclei, in part colocalized with vasopressin (VP) and oxytocin (OT). Here we subjected intact adult male rats to chronic osmotic stress to determine effects on distribution of CBG in VP and OT neurons and in neurons expressing corticotropin- releasing hormone (CRH). Drinking 2% NaCl solution for seven days resulted in increased CBG-immunoreactivity in magnocellular neurons. Triple immunofluorescence revealed increased colocalization with either VP, OT or CRH. Colocalization of CRH with VP was found only in a small portion of parvocellular neurons in the PVN. Most of the CBG-immunostained neurons within the magnocellular nuclei were devoid of CRH-immunoreactivity. Increased numbers of axons with colocalization of CBG and VP or OT were found in the internal zone of the median eminence (ME) of osmotically challenged rats. The external zone of the ME showed numerous CRH-positive neuronal projections. A small portion of them contained also CBG-immunofluorescence in both experimental animals and controls. Immunoassays of cerebrospinal fluid showed increased levels of CBG in osmotically stressed animals.Our observations suggest that hypothalamic CBG expression is malleable to functional status and that coexpression with the magnocellular peptide hormones may be of significance for endocrine stress response.

Advanced genetic and viral methods for labelling and manipulation of oxytocin and vasopressin neurones in rats.

Rats have been widely used as one of the most common laboratory animals for biological research, because their physiology, pathology, and behavioral characteristics are highly similar to humans. Recent developments in rat genetic modification techniques have now led to further their utility for a broad range of research questions, including the ability to specifically label individual neurones, and even manipulate neuronal function in rats. We have succeeded in generating several transgenic rat lines that enable visualization of specific neurones due to their expression of fluorescently-tagged oxytocin, vasopressin, and c-fos protein. Furthermore, we have been able to generate novel transgenic rat lines in which we can activate vasopressin neurones using optogenetic and chemogenetic techniques. In this review, we will summarize the techniques of genetic modification for labeling and manipulating the specific neurones. Successful examples of generating transgenic rat lines in our lab and usefulness of these rats will also be introduced. These transgenic rat lines enable the interrogation of neuronal function and physiology in a way that was not possible in the past, providing novel insights into neuronal mechanisms both in vivo and ex vivo.

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.

Aldosterone Mediated Regulation of Epithelial Sodium Channel (ENaC) Subunits in the Rat Hypothalamus

Current evidence suggests that the epithelial Na+ channel (ENaC) in the brain plays a significant role in the development of hypertension. ENaC is present in vasopressin (VP) neurons in the hypothalamus, suggesting that ENaC in VP neurons is involved in the regulation of blood pressure. Our recent study demonstrated that high dietary salt intake caused an increase in the expression and activity of ENaC that were responsible for the more depolarized basal membrane potential in VP neurons. A known regulator of ENaC expression, the mineralocorticoid receptor (MR), is present in VP neurons, suggesting that ENaC expression in VP neurons is regulated by aldosterone. In this study, the effects of aldosterone and corticosterone on ENaC were examined in acute hypothalamic slices. Real-time PCR and Western blot analysis showed that aldosterone and corticosterone treatment resulted in a significant increase in the expression of γENaC, but not α- or βENaC, and that this expression was attenuated by MR and glucocorticoid receptor (GR) antagonists. Moreover, chromatin immunoprecipitation demonstrated that the aldosterone-MR complex directly interacts with the promoter region of the γENaC gene. However, the treatment with aldosterone did not cause subcellular translocation of ENaC toward the plasma membrane nor an increase in ENaC Na+-leak current. These results indicate that expression of γENaC in VP neurons is induced by aldosterone and corticosterone through their MR and GR, respectively; however, aldosterone or corticosterone alone is not sufficient enough to increase ENaC current when they are applied to hypothalamic slices in vitro.

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.

Application of pluripotent stem cells for treatment of human neuroendocrine disorders.

The neuroendocrine system is composed of many types of functional cells. Matured cells are generally irreversible to progenitor cells and it is difficult to obtain enough from our body. Therefore, studying specific subtypes of human neuroendocrine cells in vitro has not been feasible. One of the solutions is pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. These are unlimited sources and, in theory, are able to give rise to all cell types of our body. Therefore, we can use them for regenerative medicine, developmental basic research and disease modeling. Based on this idea, differentiation methods have been studied for years. Recent studies have successfully induced hypothalamic-like progenitors from mouse and human ES/iPS cells. The induced hypothalamic-like progenitors generated hypothalamic neurons, for instance, vasopressin neurons. Induction to adenohypophysis was also reported in the manner of self-formation by three-dimensional floating cultures. Rathke's pouch-like structures, i.e., pituitary anlage, were self-organized in accordance with pituitary development in embryo. Pituitary hormone-producing cells were subsequently differentiated. The induced corticotrophs secreted adrenocorticotropic hormone in response to corticotropin-releasing hormone. When engrafted in vivo, these cells rescued systemic glucocorticoid levels in hypopituitary mice. These culture methods were characterized by replication of stepwise embryonic differentiation. It is based on the idea of mimicking the molecular environment of embryogenesis. Thanks to these improvements, these days, we can generate hormone-secreting neuroendocrine cells from pluripotent stem cells. The next problems that need to be solved are improving differentiation efficiency even further and structuring networks.

A switch from GABA inhibition to excitation of vasopressin neurons exacerbates the development angiotensin II-dependent hypertension.

Hypothalamic magnocellular neurons secrete vasopressin into the systemic circulation to maintain blood pressure by increasing renal water reabsorption and by vasoconstriction. When blood pressure rises, baroreflex activation normally inhibits vasopressin neurons via activation of GABAergic inputs. However, plasma vasopressin levels are paradoxically elevated in several models of hypertension and in some patients with essential hypertension, despite increased blood pressure. We have previously shown that vasopressin neuron activity is increased early in the development of moderate angiotensin II-dependent hypertension via blunted baroreflex inhibition of vasopressin neurons. Here, we show that antagonism of vasopressin-induced vasoconstriction slows the development of hypertension and that local administration of a GABAA receptor antagonist inhibits vasopressin neurons during, but not before, the onset of hypertension. Taken together, our data suggest that vasopressin exacerbates the increase in blood pressure evident early in the development hypertension and that blunted baroreflex inhibition of vasopressin neurons is underpinned by an excitatory shift in their response to endogenous GABA signalling. This article is protected by copyright. All rights reserved.

Activation of organum vasculosum neurons and water intake in mice by vasopressin neurons in the suprachiasmatic nucleus.

Previous studies have shown that mice housed under 12:12 h light-dark conditions display a pronounced increase in water intake during a 2-hour anticipatory period (AP) near the end of their active period (Zeitgeber Time ZT; ZT21.5-ZT23.5) compared to the preceding basal period (BP, ZT19.5-ZT21.5). This increased water intake during the AP is not associated with physiological stimuli for thirst, such as food intake, hyperosmolality, hyperthermia, or hypovolemia. Denying mice the water intake supplement during the AP causes them to be dehydrated at wake time. These observations suggest that this form of thirst may be driven by the circadian clock and serve to mitigate the dehydrating effect of absence of water intake during sleep. Here we review recent findings showing that this behavior is mediated by vasopressin (VP) containing neurons in the suprachiasmatic nucleus (SCN). SCN VP neurons project to the organum vasculosum lamina terminalis (OVLT) where the activity dependent release of VP causes excitation of thirst-promoting neurons. SCN VP neurons increase their electrical activity during the AP and the resultant release of VP causes an increase in the action potential firing rate of OVLT neurons. Experiments involving optogenetic control of VP release from the axon terminals of SCN neurons indicate that this network mechanism is necessary and sufficient to mediate pre-sleep water intake in mice. These findings provide insight into the output mechanisms that are used by the central clock to generate circadian rhythms, and reveal that the regulation of water intake contributes to osmoregulatory homeostasis during sleep. This article is protected by copyright. All rights reserved.

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, β, 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 γ ENaC expression and exhibited proteolytic cleavage of this subunit compared to Sprague–Dawley (SD) rats. Additionally, Dahl‐SS rats had dense somato‐dendritic γ ENaC immunoreactivity in VP neurons, which was absent in SD rats. In contrast, SD rats fed a high salt diet had significantly decreased α ENaC 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.

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

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

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

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 μM Cd2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 μM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.