Thirst recruits phasic dopamine signaling through subfornical organ neurons.

Thirst is a highly potent drive that motivates organisms to seek out and consume balance-restoring stimuli. The detection of dehydration is well understood and involves signals of peripheral origin and the sampling of internal milieu by first order homeostatic neurons within the lamina terminalis-particularly glutamatergic neurons of the subfornical organ expressing CaMKIIa (SFOCaMKIIa). However, it remains unknown whether mesolimbic dopamine pathways that are critical for motivation and reinforcement integrate information from these "early" dehydration signals. We used in vivo fiber photometry in the ventral tegmental area and measured phasic dopamine responses to a water-predictive cue. Thirst, but not hunger, potentiated the phasic dopamine response to the water cue. In euvolemic rats, the dipsogenic hormone angiotensin II, but not the orexigenic hormone ghrelin, potentiated the dopamine response similarly to that observed in water-deprived rats. Chemogenetic manipulations of SFOCaMKIIa revealed bidirectional control of phasic dopamine signaling during cued water reward. Taking advantage of within-subject designs, we found predictive relationships between changes in cue-evoked dopamine response and changes in behavioral responses-supporting a role for dopamine in motivation induced by homeostatic need. Collectively, we reveal a putative mechanism for the invigoration of goal-directed behavior: internal milieu communicates to first order, need state-selective circuits to potentiate the mesolimbic dopamine system's response to cues predictive of restorative stimuli.

The subfornical organ regulates acidosis-evoked fear by engaging microglial acid-sensor TDAG8 and forebrain neurocircuits in male mice.

An important role of pH homeostasis has been suggested in the physiology of panic disorder, with acidosis as an interoceptive trigger leading to fear and panic. Identification of novel mechanisms that can translate acidosis into fear will promote a better understanding of panic physiology. The current study explores a role of the subfornical organ (SFO), a blood-brain barrier compromised brain area, in translating acidosis to fear-relevant behaviors. We performed SFO-targeted acidification in male, wild-type mice and mice lacking microglial acid-sensing G protein-coupled receptor-T-cell death-associated gene 8 (TDAG8). Localized SFO acidification evoked significant freezing and reduced exploration that was dependent on the presence of acid-sensor TDAG8. Acidosis promoted the activation of SFO microglia and neurons that were absent in TDAG8-deficient mice. The assessment of regional neuronal activation in wild-type and TDAG8-deficient mice following SFO acidification revealed significant acidosis and genotype-dependent alterations in the hypothalamus, amygdala, prefrontal cortex, and periaqueductal gray nuclei. Furthermore, mapping of interregional co-activation patterns revealed that SFO acidosis promoted positive hypothalamic-cortex associations and desynchronized SFO-cortex and amygdala-cortex associations, suggesting an interplay of homeostatic and fear regulatory areas. Importantly, these alterations were not evident in TDAG8-deficient mice. Overall, our data support a regulatory role of subfornical organ microglial acid sensing in acidosis-evoked fear, highlighting a centralized role of blood-brain barrier compromised nodes in interoceptive sensing and behavioral regulation. Identification of pathways by which humoral information can modulate fear behavior is relevant to panic disorder, where aberrant interoceptive signaling has been reported.

Subfornical organ interleukin 1 receptor: A novel regulator of spontaneous and conditioned fear associated behaviors in mice.

Impaired threat responding and fear regulation is a hallmark of psychiatric conditions such as post-traumatic stress disorder (PTSD) and Panic Disorder (PD). Most studies have focused on external psychogenic threats to study fear, however, accumulating evidence suggests a primary role of homeostatic perturbations and interoception in regulating emotional behaviors. Heightened reactivity to interoceptive threat carbon dioxide (CO2) inhalation associates with increased risk for developing PD and PTSD, however, contributory mechanisms and molecular targets are not well understood. Previous studies from our group suggested a potential role of interleukin 1 receptor (IL-1R1) signaling within BBB-devoid sensory circumventricular organ, the subfornical organ (SFO) in CO2-evoked fear. However, the necessity of SFO-IL-1R1 in regulating CO2-associated spontaneous fear as well as, long-term fear potentiation relevant to PD/PTSD has not been investigated. The current study tested male mice with SFO-targeted microinfusion of the IL-1R1 antagonist (IL-1RA) or vehicle in a recently developed CO2-startle-fear conditioning-extinction paradigm. Consistent with our hypothesis, SFO IL-1RA treatment elicited significant attenuation of freezing and increased rearing during CO2 inhalation suggesting SFO-IL1R1 regulation of spontaneous fear to CO2. Intriguingly, SFO IL-1RA treatment normalized CO2-associated potentiation of conditioned fear and impaired extinction a week later suggesting modulation of long-term fear by SFO-IL-1R1 signaling. Post behavior FosB mapping revealed recruitment of prefrontal cortex-amygdala-periaqueductal gray (PAG) areas in SFO-IL-1RA mediated effects. Additionally, we localized cellular IL-1R1 expression within the SFO to blood vessel endothelial cells and observed CO2-induced alterations in IL-1ß/IL-1R1 expression in peripheral mononuclear cells and SFO. Lastly, CO2-evoked microglial activation was attenuated in SFO-IL-1RA treated mice. These observations suggest a peripheral monocyte-endothelial-microglia interplay in SFO-IL-1R1 modulation of CO2-associated spontaneous fear and delayed fear memory. Collectively, our data highlight a novel, "bottom-up" neuroimmune mechanism that integrates interoceptive and exteroceptive threat processing of relevance to fear-related pathologies.

Sex- and age-dependent differences in the hormone and drinking responses to water deprivation.

Maintenance of the volume and osmolality of body fluids is important, and the adaptive responses recruited to protect against osmotic stress are crucial for survival. The objective of this work was to compare the responses that occur in aging male and female rats during water deprivation. For this purpose, groups of male and female Wistar rats aged 3 mo (adults) or 18 mo (old) were submitted to water deprivation (WD) for 48 h. The water and sodium (0.15 M NaCl) intake, plasma concentrations of oxytocin (OT), arginine vasopressin (AVP), corticosterone (CORT), atrial natriuretic peptide (ANP), and angiotensin II (ANG II) were determined in hydrated and water-deprived animals. In response to WD, old male and female rats drank less water and saline than adults, and both adult and old females drank more water and saline than respective males. Dehydrated old animals displayed lower ANG II plasma concentration and CORT response compared with the respective normohydrated rats. Dehydrated adult males had higher plasma ANP and AVP as well as lower CORT concentrations than dehydrated adult females. Moreover, plasma OT and CORT levels of old female rats were higher than those in the dehydrated old male rats. Relative expression of ANG II type 1 receptor mRNA was decreased in the subfornical organ of adult and old male rats as well as adult female rats in response to WD. In conclusion, the study elucidated the effect of sex and age on responses induced by WD, altering the degree of dehydration induced by 48 h of WD.

Subfornical organ insulin receptors tonically modulate cardiovascular and metabolic function.

Insulin acts within the central nervous system through the insulin receptor to influence both metabolic and cardiovascular physiology. While a major focus has been placed on hypothalamic regions, participation of extrahypothalamic insulin receptors in cardiometabolic regulation remains largely unknown. We hypothesized that insulin receptors in the subfornical organ (SFO), a forebrain circumventricular region devoid of a blood-brain barrier, are involved in metabolic and cardiovascular regulation. Immunohistochemistry in mice revealed widespread insulin receptor-positive cells throughout the rostral to caudal extent of the SFO. SFO-targeted adenoviral delivery of Cre-recombinase in insulin receptorlox/lox mice resulted in sufficient ablation of insulin receptors in the SFO. Interestingly, when mice were maintained on a normal chow diet, deletion of SFO insulin receptors resulted in greater weight gain and adiposity, relative to controls, independently of changes in food intake. In line with this, ablation of insulin receptors in the SFO was associated with marked hepatic steatosis and hypertriglyceridemia. Selective removal of SFO insulin receptors also resulted in a lower mean arterial blood pressure, which was primarily due to a reduction in diastolic blood pressure, whereas systolic blood pressure remained unchanged. Cre-mediated targeting of SFO insulin receptors did not influence heart rate. These data demonstrate multidirectional roles for insulin receptor signaling in the SFO, with ablation of SFO insulin receptors resulting in an overall deleterious metabolic state while at the same time maintaining blood pressure at low levels. These novel findings further suggest that alterations in insulin receptor signaling in the SFO could contribute to metabolic syndrome phenotypes.

Ionic mechanisms underlying tonic and burst firing behavior in subfornical organ neurons: a combined experimental and modeling study.

Subfornical organ (SFO) neurons exhibit heterogeneity in current expression and spiking behavior, where the two major spiking phenotypes appear as tonic and burst firing. Insight into the mechanisms behind this heterogeneity is critical for understanding how the SFO, a sensory circumventricular organ, integrates and selectively influences physiological function. To integrate efficient methods for studying this heterogeneity, we built a single-compartment, Hodgkin-Huxley-type model of an SFO neuron that is parameterized by SFO-specific in vitro patch-clamp data. The model accounts for the membrane potential distribution and spike train variability of both tonic and burst firing SFO neurons. Analysis of model dynamics confirms that a persistent Na+ and Ca2+ currents are required for burst initiation and maintenance and suggests that a slow-activating K+ current may be responsible for burst termination in SFO neurons. Additionally, the model suggests that heterogeneity in current expression and subsequent influence on spike afterpotential underlie the behavioral differences between tonic and burst firing SFO neurons. Future use of this model in coordination with single neuron patch-clamp electrophysiology provides a platform for explaining and predicting the response of SFO neurons to various combinations of circulating signals, thus elucidating the mechanisms underlying physiological signal integration within the SFO. NEW & NOTEWORTHY Our understanding of how the subfornical organ (SFO) selectively influences autonomic nervous system function remains incomplete but theoretically results from the electrical responses of SFO neurons to physiologically important signals. We have built a computational model of SFO neurons, derived from and supported by experimental data, which explains how SFO neurons produce different electrical patterns. The model provides an efficient system to theoretically and experimentally explore how changes in the essential features of SFO neurons affect their electrical activity.

Tumor necrosis factor-α potentiates the effects of angiotensin II on subfornical organ neurons.

Inflammation is thought to play a fundamental role in the pathophysiology of hypertension and heart failure, although the mechanisms for this remain unclear. Proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), influence the subfornical organ (SFO) to modulate sympathetic activity and blood pressure. The pressor effects of TNF-α in the SFO are partially mediated by angiotensin II (ANG II) receptor type 1 (AT1R), and TNF-α is known to potentiate ANG II-induced hypertension. However, the cellular mechanism of the interaction between TNF-α and ANG II/AT1R signaling remains unknown. In the present study, we performed Ca2+ imaging on dissociated SFO neurons in vitro from male Sprague-Dawley rats to determine whether TNF-α modulates ANG II-induced increases in intracellular Ca2+ in SFO neurons. We first established that a proportion of SFO neurons respond to ANG II, an effect that required AT1R signaling and extracellular Ca2+. We then tested the hypothesis that TNF-α may modulate the effects of ANG II on SFO neurons by examining the effects of TNF-α treatment on the ANG II-induced rise in intracellular Ca2+. We discovered that TNF-α potentiated the ANG II-induced rise in intracellular Ca2+, an effect that was dependent on the duration of TNF-α treatment. Finally, we determined that this potentiation of ANG II-induced Ca2+ activity relied on tetrodotoxin-sensitive voltage-gated Na+ (vgNa+) channels. These data suggest that the potentiation of ANG II/AT1R activity by TNF-α in SFO neurons results from the previously demonstrated ability of this cytokine to modulate the activation threshold of vgNa+ currents.

Evidence for intraventricular secretion of angiotensinogen and angiotensin by the subfornical organ using transgenic mice.

Direct intracerebroventricular injection of angiotensin II (ANG II) causes increases in blood pressure and salt and water intake, presumably mimicking an effect mediated by an endogenous mechanism. The subfornical organ (SFO) is a potential source of cerebrospinal fluid (CSF), ANG I, and ANG II, and thus we hypothesized that the SFO has a secretory function. Endogenous levels of angiotensinogen (AGT) and renin are very low in the brain. We therefore examined the immunohistochemical localization of angiotensin peptides and AGT in the SFO, and AGT in the CSF in two transgenic models that overexpress either human AGT (A+ mice), or both human AGT (hAGT) and human renin (SRA mice) in the brain. Measurements were made at baseline and following volumetric depletion of CSF. Ultrastructural analysis with immunoelectron microscopy revealed that superficially located ANG I/ANG II and AGT immunoreactive cells in the SFO were vacuolated and opened directly into the ventricle. Withdrawal of CSF produced an increase in AGT in the CSF that was accompanied by a large decline in AGT immunoreactivity within SFO cells. Our data provide support for the hypothesis that the SFO is a secretory organ that releases AGT and possibly ANG I/ANG II into the ventricle at least under conditions when genes that control the renin-angiotensin system are overexpressed in mice.

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.

A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei.

Physiological roles for the subfornical organ: a dynamic transcriptome shaped by autonomic state.

The subfornical organ (SFO) is a circumventricular organ recognized for its ability to sense and integrate hydromineral and hormonal circulating fluid balance signals, information which is transmitted to central autonomic nuclei to which SFO neurons project. While the role of SFO was once synonymous with physiological responses to osmotic, volumetric and cardiovascular challenge, recent data suggest that SFO neurons also sense and integrate information from circulating signals of metabolic status. Using microarrays, we have confirmed the expression of receptors already described in the SFO, and identified many novel transcripts expressed in this circumventricular organ including receptors for many of the critical circulating energy balance signals such as adiponectin, apelin, endocannabinoids, leptin, insulin and peptide YY. This transcriptome analysis also identified SFO transcripts, the expressions of which are significantly changed by either 72 h dehydration, or 48 h starvation, compared to fed and euhydrated controls. Expression and potential roles for many of these targets are yet to be confirmed and elucidated. Subsequent validation of data for adiponectin and leptin receptors confirmed that receptors for both are expressed in the SFO, that discrete populations of neurons in this tissue are functionally responsive to these adipokines, and that such responsiveness is regulated by physiological state. Thus, transcriptomic analysis offers great promise for understanding the integrative complexity of these physiological systems, especially with development of technologies allowing description of the entire transcriptome of single, carefully phenotyped, SFO neurons. These data will ultimately elucidate mechanisms through which these uniquely positioned neurons respond to and integrate complex circulating signals.

Deletion of proton-sensing receptor GPR4 associates with lower blood pressure and lower binding of angiotensin II receptor in SFO.

Diets rich in grains and meat and low in fruits and vegetables (acid-producing diets) associate with incident hypertension, whereas vegetarian diets associate with lower blood pressure (BP). However, the pathways that sense and mediate the effects of acid-producing diets on BP are unknown. Here, we examined the impact of the deletion of an acid sensor GPR4 on BP. GPR4 is a proton-sensing G protein-coupled receptor and an acid sensor in brain, kidney, and blood vessels. We found that GPR4 mRNA was higher in subfornical organ (SFO) than other brain regions. GPR4 protein was abundant in SFO and present in capillaries throughout the brain. Since SFO partakes in BP regulation through the renin-angiotensin system (RAS), we measured BP in GPR4-/- and GPR4+/+ mice and found that GPR4 deletion associated with lower systolic BP: 87 ± 1 mmHg in GPR4-/- (n = 35) vs. 99 ± 2 mmHg (n = 29) in GPR4+/+; P < 0.0001, irrespective of age and sex. Angiotensin II receptors detected by 125I-Sarthran binding were lower in GPR4-/- than GPR4+/+ mice in SFO and in paraventricular nucleus of hypothalamus. Circulating angiotensin peptides were comparable in GPR4-/- and GPR4+/+ mice, as were water intake and excretion, serum and urine osmolality, and fractional excretion of sodium, potassium, or chloride. A mild metabolic acidosis present in GPR4-/- mice did not associate with elevated BP, implying that deficiency of GPR4 may preclude the effect of chronic acidosis on BP. Collectively, these results posit the acid sensor GPR4 as a novel component of central BP control through interactions with the RAS.

Endoplasmic reticulum stress increases brain MAPK signaling, inflammation and renin-angiotensin system activity and sympathetic nerve activity in heart failure.

We previously reported that endoplasmic reticulum (ER) stress is induced in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN) of heart failure (HF) rats and is reduced by inhibition of mitogen-activated protein kinase (MAPK) signaling. The present study further examined the relationship between brain MAPK signaling, ER stress, and sympathetic excitation in HF. Sham-operated (Sham) and HF rats received a 4-wk intracerebroventricular (ICV) infusion of vehicle (Veh) or the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 10 µg/day). Lower mRNA levels of the ER stress biomarkers GRP78, ATF6, ATF4, and XBP-1s in the SFO and PVN of TUDCA-treated HF rats validated the efficacy of the TUDCA dose. The elevated levels of phosphorylated p44/42 and p38 MAPK in SFO and PVN of Veh-treated HF rats, compared with Sham rats, were significantly reduced in TUDCA-treated HF rats as shown by Western blot and immunofluorescent staining. Plasma norepinephrine levels were higher in Veh-treated HF rats, compared with Veh-treated Sham rats, and were significantly lower in the TUDCA-treated HF rats. TUDCA-treated HF rats also had lower mRNA levels for angiotensin converting enzyme, angiotensin II type 1 receptor, tumor necrosis factor-α, interleukin-1ß, cyclooxygenase-2, and NF-κB p65, and a higher mRNA level of IκB-α, in the SFO and PVN than Veh-treated HF rats. These data suggest that ER stress contributes to the augmented sympathetic activity in HF by inducing MAPK signaling, thereby promoting inflammation and renin-angiotensin system activity in key cardiovascular regulatory regions of the brain.

Salt and gene expression: evidence for [Na+]i/[K+]i-mediated signaling pathways.

Our review focuses on the recent data showing that gene transcription and translation are under the control of signaling pathways triggered by modulation of the intracellular sodium/potassium ratio ([Na+]i/[K+]i). Side-by-side with sensing of osmolality elevation by tonicity enhancer-binding protein (TonEBP, NFAT5), [Na+]i/[K+]i-mediated excitation-transcription coupling may contribute to the transcriptomic changes evoked by high salt consumption. This novel mechanism includes the sensing of heightened Na+ concentration in the plasma, interstitial, and cerebrospinal fluids via augmented Na+ influx in the endothelium, immune system cells, and the subfornical organ, respectively. In these cells, [Na+]i/[K+]i ratio elevation, triggered by augmented Na+ influx, is further potentiated by increased production of endogenous Na+,K+-ATPase inhibitors documented in salt-sensitive hypertension.

Sodium sensing in the brain.

Sodium (Na) homeostasis is crucial for life, and the Na(+) level ([Na(+)]) of body fluids is strictly maintained at a range of 135-145 mM. However, the existence of a [Na(+)] sensor in the brain has long been controversial until Nax was identified as the molecular entity of the sensor. This review provides an overview of the [Na(+)]-sensing mechanism in the brain for the regulation of salt intake by summarizing a series of our studies on Nax. Nax is a Na channel expressed in the circumventricular organs (CVOs) in the brain. Among the CVOs, the subfornical organ (SFO) is the principal site for the control of salt intake behavior, where Nax populates the cellular processes of astrocytes and ependymal cells enveloping neurons. A local expression of endothelin-3 in the SFO modulates the [Na(+)] sensitivity for Nax activation, and thereby Nax is likely to be activated in the physiological [Na(+)] range. Nax stably interacts with Na(+)/K(+)-ATPase whereby Na(+) influx via Nax is coupled with activation of Na(+)/K(+)-ATPase associated with the consumption of ATP. The consequent activation of anaerobic glucose metabolism of Nax-positive glial cells upregulates the cellular release of lactate, and this lactate functions as a gliotransmitter to activate GABAergic neurons in the SFO. The GABAergic neurons presumably regulate hypothetic neurons involved in the control of salt intake behavior. Recently, a patient with essential hypernatremia caused by autoimmunity to Nax was found. In this case, the hypernatremia was considered to be induced by the complement-mediated cell death in the CVOs, where Nax specifically populates.

Actions of a hydrogen sulfide donor (NaHS) on transient sodium, persistent sodium, and voltage-gated calcium currents in neurons of the subfornical organ.

Hydrogen sulfide (H2S) is an endogenously found gasotransmitter that has been implicated in a variety of beneficial physiological functions. This study was performed to investigate the cellular mechanisms underlying actions of H2S previously observed in subfornical organ (SFO), where H2S acts to regulate blood pressure through a depolarization of the membrane and an overall increase in the excitability of SFO neurons. We used whole cell patch-clamp electrophysiology in the voltage-clamp configuration to analyze the effect of 1 mM NaHS, an H2S donor, on voltage-gated potassium, sodium, and calcium currents. We observed no effect of NaHS on potassium currents; however, both voltage-gated sodium currents (persistent and transient) and the N-type calcium current had a depolarized activation curve and an enhanced peak-induced current in response to a series of voltage-step and ramp protocols run in the control and NaHS conditions. These effects were not responsible for the previously observed depolarization of the membrane potential, as depolarizing effects of H2S were still observed following block of these conductances with tetrodotoxin (5 µM) and ω-conotoxin-GVIA (100 nM). Our studies are the first to investigate the effect of H2S on a variety of voltage-gated conductances in a single brain area, and although they do not explain mechanisms underlying the depolarizing actions of H2S on SFO neurons, they provide evidence of potential mechanisms through which this gasotransmitter influences the excitability of neurons in this important brain area as a consequence of the modulation of multiple ion channels.

Angiotensin II type 1a receptors in subfornical organ contribute towards chronic intermittent hypoxia-associated sustained increase in mean arterial pressure.

Sleep apnea is associated with hypertension. The mechanisms contributing to a sustained increase in mean arterial pressure (MAP) even during normoxic awake-state remain unknown. Rats exposed to chronic intermittent hypoxia for 7 days, a model of the hypoxemia associated with sleep apnea, exhibit sustained increases in MAP even during the normoxic dark phase. Activation of the renin-angiotensin system (RAS) has been implicated in chronic intermittent hypoxia (CIH) hypertension. Since the subfornical organ (SFO) serves as a primary target for the central actions of circulating ANG II, we tested the effects of ANG II type 1a receptor (AT1aR) knockdown in the SFO on the sustained increase in MAP in this CIH model. Adeno-associated virus carrying green fluorescent protein (GFP) and small-hairpin RNA against either AT1aR or a scrambled control sequence (SCM) was stereotaxically injected in the SFO of rats. After recovery, MAP, heart rate, respiratory rate, and activity were continuously recorded using radiotelemetry. In the normoxic groups, the recorded variables did not deviate from the baseline values. Both CIH groups exhibited significant increases in MAP during CIH exposures (P < 0.05). During the normoxic dark phase in the CIH groups, only the SCM-injected group exhibited a sustained increase in MAP (P < 0.05). The AT1aR-CIH group showed significant decreases in FosB/ΔFosB staining in the median preoptic nucleus and the paraventricular nuclei of the hypothalamus compared with the SCM-CIH group. Our data indicate that AT1aRs in the SFO are critical for the sustained elevation in MAP and increased FosB/ΔFosB expression in forebrain autonomic nuclei associated with CIH.

Mechanisms of brain renin angiotensin system-induced drinking and blood pressure: importance of the subfornical organ.

It is critical for cells to maintain a homeostatic balance of water and electrolytes because disturbances can disrupt cellular function, which can lead to profound effects on the physiology of an organism. Dehydration can be classified as either intra- or extracellular, and different mechanisms have developed to restore homeostasis in response to each. Whereas the renin-angiotensin system (RAS) is important for restoring homeostasis after dehydration, the pathways mediating the responses to intra- and extracellular dehydration may differ. Thirst responses mediated through the angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptors (AT2R) respond to extracellular dehydration and intracellular dehydration, respectively. Intracellular signaling factors, such as protein kinase C (PKC), reactive oxygen species (ROS), and the mitogen-activated protein (MAP) kinase pathway, mediate the effects of central angiotensin II (ANG II). Experimental evidence also demonstrates the importance of the subfornical organ (SFO) in mediating some of the fluid intake effects of central ANG II. The purpose of this review is to highlight the importance of the SFO in mediating fluid intake responses to dehydration and ANG II.

Genetic knockdown of estrogen receptor-alpha in the subfornical organ augments ANG II-induced hypertension in female mice.

The present study tested the hypotheses that 1) ERα in the brain plays a key role in the estrogen-protective effects against ANG II-induced hypertension, and 2) that the subfornical organ (SFO) is a key site where ERα mediates these protective actions. In this study, a "floxed" ERα transgenic mouse line (ERα(flox)) was used to create models in which ERα was knocked down in the brain or just in the SFO. Female mice with ERα ablated in the nervous system (Nestin-ERα(-) mice) showed greater increases in blood pressure (BP) in response to ANG II. Furthermore, females with ERα knockdown specifically in the SFO [SFO adenovirus-Cre (Ad-Cre) injected ERα(flox) mice] also showed an enhanced pressor response to ANG II. Immunohistochemical (IHC), RT-PCR, and Western blot analyses revealed a marked reduction in the expression of ERα in nervous tissues and, in particular, in the SFO. These changes were not present in peripheral tissues in Nestin-ERα(-) mice or Ad-Cre-injected ERα(flox) mice. mRNA expression of components of the renin-angiotensin system in the lamina terminalis were upregulated in Nestin-ERα(-) mice. Moreover, ganglionic blockade on day 7 after ANG II infusions resulted in a greater reduction of BP in Nestin-ERα(-) mice or SFO Ad-Cre-injected mice, suggesting that knockdown of ERα in the nervous system or the SFO alone augments central ANG II-induced increase in sympathetic tone. The results indicate that interfering with the action of estrogen on SFO ERα is sufficient to abolish the protective effects of estrogen against ANG II-induced hypertension.

Relaxin increases sympathetic nerve activity and activates spinally projecting neurons in the paraventricular nucleus of nonpregnant, but not pregnant, rats.

Pregnancy is characterized by increased blood volume and baseline sympathetic nerve activity (SNA), vasodilation, and tachycardia. Relaxin (RLX), an ovarian hormone elevated in pregnancy, activates forebrain sites involved in control of blood volume and SNA through ANG II-dependent mechanisms and contributes to adaptations during pregnancy. In anesthetized, arterial baroreceptor-denervated nonpregnant (NP) rats, RLX microinjected into the subfornical organ (SFO; 0.77 pmol in 50 nl) produced sustained increases in lumbar SNA (8 ± 3%) and mean arterial pressure (MAP; 26 ± 4 mmHg). Low-dose intracarotid artery infusion of RLX (155 pmol·ml(-1)·h(-1); 1.5 h) had minor transient effects on AP and activated neurons [increased Fos-immunoreactivity (IR)] in the SFO and in spinally projecting (19 ± 2%) and arginine-vasopressin (AVP)-IR (21 ± 5%) cells in the paraventricular nucleus of the hypothalamus of NP, but not pregnant (P), rats. However, mRNA for RLX and ANG II type 1a receptors in the SFO was preserved in pregnancy. RLX receptor-IR is present in the region of the SFO in NP and P rats and is localized in astrocytes, the major source of angiotensinogen in the SFO. These data provide an anatomical substrate for a role of RLX in the resetting of AVP secretion and increased baseline SNA in pregnancy. Since RLX and ANG II receptor expression was preserved in the SFO of P rats, we speculate that the lack of response to exogenous RLX may be due to maximal activation by elevated endogenous levels of RLX in near-term pregnancy.