Circulating mitochondria promoted endothelial cGAS-derived neuroinflammation in subfornical organ to aggravate sympathetic overdrive in heart failure mice.

BACKGROUND: Sympathoexcitation contributes to myocardial remodeling in heart failure (HF). Increased circulating pro-inflammatory mediators directly act on the Subfornical organ (SFO), the cardiovascular autonomic center, to increase sympathetic outflow. Circulating mitochondria (C-Mito) are the novel discovered mediators for inter-organ communication. Cyclic GMP-AMP synthase (cGAS) is the pro-inflammatory sensor of damaged mitochondria. OBJECTIVES: This study aimed to assess the sympathoexcitation effect of C-Mito in HF mice via promoting endothelial cGAS-derived neuroinflammation in the SFO. METHODS: C-Mito were isolated from HF mice established by isoprenaline (0.0125 mg/kg) infusion via osmotic mini-pumps for 2 weeks. Structural and functional analyses of C-Mito were conducted. Pre-stained C-Mito were intravenously injected every day for 2 weeks. Specific cGAS knockdown (cGAS KD) in the SFO endothelial cells (ECs) was achieved via the administration of AAV9-TIE-shRNA (cGAS) into the SFO. The activation of cGAS in the SFO ECs was assessed. The expression of the mitochondrial redox regulator Dihydroorotate dehydrogenase (DHODH) and its interaction with cGAS were also explored. Neuroinflammation and neuronal activation in the SFO were evaluated. Sympathetic activity, myocardial remodeling, and cardiac systolic dysfunction were measured. RESULTS: C-Mito were successfully isolated, which showed typical structural characteristics of mitochondria with double-membrane and inner crista. Further analysis showed impaired respiratory complexes activities of C-Mito from HF mice (C-MitoHF) accompanied by oxidative damage. C-Mito entered ECs, instead of glial cells and neurons in the SFO of HF mice. C-MitoHF increased the level of ROS and cytosolic free double-strand DNA (dsDNA), and activated cGAS in cultured brain endothelial cells. Furthermore, C-MitoHF highly expressed DHODH, which interacted with cGAS to facilitate endothelial cGAS activation. C-MitoHF aggravated endothelial inflammation, microglial/astroglial activation, and neuronal sensitization in the SFO of HF mice, which could be ameliorated by cGAS KD in the ECs of the SFO. Further analysis showed C-MitoHF failed to exacerbate sympathoexcitation and myocardial sympathetic hyperinnervation in cGAS KD HF mice. C-MitoHF promoted myocardial fibrosis and hypertrophy, and cardiac systolic dysfunction in HF mice, which could be ameliorated by cGAS KD. CONCLUSION: Collectively, we demonstrated that damaged C-MitoHF highly expressed DHODH, which promoted endothelial cGAS activation in the SFO, hence aggravating the sympathoexcitation and myocardial injury in HF mice, suggesting that C-Mito might be the novel therapeutic target for sympathoexcitation in HF.

Autoimmunity related to anti-Nax and anti-ZSCAN1 autoantibodies in adipsic hypernatremia.

"Adipsic hypernatremia" is clinically characterized by chronic elevation of plasma [Na+] with an inappropriate lack of thirst and upward resetting of the osmotic set point for arginine vasopressin (AVP) secretion, combined with a relative deficiency of AVP, thereby resulting in persistent hypernatremia. Many cases are accompanied by structural lesions in the hypothalamus, pituitary gland, and circumventricular organs (CVOs). On the other hand, cases without structural lesions have been reported since the 1970s, but the pathophysiology was unknown for a long time. In 2010, Hiyama et al. reported that an anti-Nax antibody response caused adipsic hypernatremia in a pediatric case with ganglioblastoma. In recent years, advances in clinical research have led researchers to recognize that an autoimmunological pathogenic mechanism might be associated with periventricular organs such as the subfornical organ (SFO). In addition, in pediatric cases diagnosed as ROHHAD (rapid-onset obesity with hypoventilation, hypothalamic dysfunction, autonomic dysregulation) syndrome, it has been reported that half of the cases have abnormal serum Na levels, and some research findings indicated an autoimmune mechanism acting on the organs of the hypothalamus and CVOs. Then, anti-ZSCAN1 antibody response was detected in cases diagnosed as ROHHAD-NET in 2022. In this review, by summarizing a series of studies on Nax and ZSCAN1, which are expressed in the hypothalamus, pituitary gland, and SFO, I would like to describe the current findings of the autoimmune pathogenesis of adipsic hypernatremia.

Anti-ZSCAN1 Autoantibodies Are a Feasible Diagnostic Marker for ROHHAD Syndrome Not Associated with a Tumor.

Recent studies have reported the presence of autoantibodies against zinc finger and SCAN domain-containing protein 1 (ZSCAN1) in the sera of patients with rapid-onset obesity with hypoventilation, hypothalamic and autonomic dysregulation (ROHHAD) syndrome associated with neuroendocrine tumors, suggesting immunologic and paraneoplastic processes as the pathologic underpinnings. Moreover, several hypothalamic regions, including the subfornical organ (SFO), were reported to exhibit antibody reactivity in a patient with ROHHAD syndrome not associated with a tumor. Whether ROHHAD syndrome not associated with a tumor is associated with anti-ZSCAN1 autoantibodies remains unclear. We used a comprehensive protein array analysis to identify candidate molecules in the sera of patients with ROHHAD syndrome and identified ZSCAN1 as a target antigen. We also found that ZSCAN1 was co-expressed at the site of antibody reactivity to the IgG in the patient serum observed in mouse SFOs and an enzyme-linked immunosorbent assay showed that >85% of the patients with ROHHAD syndrome were positive for anti-ZSCAN1 autoantibodies. These results suggest anti-ZSCAN1 autoantibodies as a feasible diagnostic marker in ROHHAD syndrome regardless of the presence of a tumor.

Salt-induced phosphoproteomic changes in the subfornical organ in rats with chronic kidney disease.

OBJECTIVES: Subfornical organ (SFO) is vital in chronic kidney disease (CKD) progression caused by high salt levels. The current study investigated the effects of high salt on phosphoproteomic changes in SFO in CKD rats. METHODS: 5/6 nephrectomized rats were fed a normal-salt diet (0.4%) (NC group) or a high-salt diet (4%) (HC group) for three weeks, while sham-operated rats were fed a normal-salt diet (0.4%) (NS group). For phosphoproteomic analysis of SFO in different groups, TiO2 enrichment, isobaric tags for relative and absolute quantification (iTRAQ) labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used. RESULTS: There were 6808 distinct phosphopeptides found, which corresponded to 2661 phosphoproteins. NC group had 168 upregulated and 250 downregulated phosphopeptides compared to NS group. Comparison to NC group, HC group had 154 upregulated and 124 downregulated phosphopeptides. Growth associated protein 43 (GAP43) and heat shock protein 27 (Hsp27) were significantly upregulated phosphoproteins and may protect against high-salt damage. Differential phosphoproteins with tight functional connection were synapse proteins and microtubule-associated proteins, implying that high-salt diet disrupted brain's structure and function. Furthermore, differential phosphoproteins in HC/NC comparison group were annotated to participate in GABAergic synapse signaling pathway and aldosterone synthesis and secretion, which attenuated inhibitory neurotransmitter effects and increased sympathetic nerve activity (SNA). DISCUSSION: This large scale phosphoproteomic profiling of SFO sheds light on how salt aggravates CKD via the central nervous system.

Identification of clinical factors related to antibody-mediated immune response to the subfornical organ.

OBJECTIVE: We recently reported cases of adipsic hypernatremia caused by autoantibodies against the subfornical organ in patients with hypothalamic-pituitary lesions. This study aimed to clarify the clinical features of newly identified patients with adipsic hypernatremia whose sera displayed immunoreactivity to the mouse subfornical organ. DESIGN: Observational cohort study of patients diagnosed with adipsic hypernatremia in Japan, United States, and Europe. METHODS: The study included 22 patients with adipsic hypernatremia but without overt structural changes in the hypothalamic-pituitary region and congenital disease. Antibody response to the mouse subfornical organ was determined using immunohistochemistry. The clinical characteristics were compared between the patients with positive and negative antibody responses. RESULTS: Antibody response to the mouse subfornical organ was detected in the sera of 16 patients (72.7%, female/male ratio, 1:1, 12 pediatric and 4 adult patients). The prolactin levels at the time of diagnosis were significantly higher in patients with positive subfornical organ (SFO) immunoreactivity than in those with negative SFO immunoreactivity (58.9 ± 33.5 vs. 22.9 ± 13.9 ng/ml, p < .05). Hypothalamic disorders were found in 37.5% of the patients with positive SFO immunoreactivity. Moreover, six patients were diagnosed with rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation/neural tumor syndrome after the diagnosis of adipsic hypernatremia. Plasma renin activity levels were significantly higher in patients with serum immunoreactivity to the Nax channel. CONCLUSIONS: The patients with serum immunoreactivity to the SFO had higher prolactin levels and hypothalamic disorders compared to those without the immunoreactivity. The clinical characteristics of patients with serum immunoreactivity to the subfornical organ included higher prolactin levels and hypothalamic disorders, which were frequently associated with central hypothyroidism and the presence of retroperitoneal tumors.

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.

Autoimmunity Related to Adipsic Hypernatremia and ROHHAD Syndrome.

Specific antibody responses to subfornical organs, including Nax antibody, have been reported in patients with adipsic hypernatremia of unknown etiology who do not have structural lesions in the hypothalamic-pituitary gland. The subfornical organ, also referred to as the window of the brain, is a sensing site that monitors sodium and osmotic pressure levels. On the other hand, ROHHAD syndrome is a rare disease for which the etiology of the hypothalamic disorder is unknown, and there have been some reports in recent years describing its association with autoimmune mechanisms. In addition, abnormal Na levels, including hypernatremia, are likely to occur in this syndrome. When comparing the clinical features of adipsic hypernatremia due to autoimmune mechanisms and ROHHAD syndrome, there are similar hypothalamic-pituitary dysfunction symptoms in addition to abnormal Na levels. Since clinical diagnoses of autoimmunological adipsic hypernatremia and ROHAD syndrome might overlap, we need to understand the essential etiology and carry out precise assessments to accurately diagnose patients and provide effective treatment. In this review, I review the literature on the autoimmune mechanism reported in recent years and describe the findings obtained so far and future directions.

TNF-α-induced sympathetic excitation requires EGFR and ERK1/2 signaling in cardiovascular regulatory regions of the forebrain.

Peripherally or centrally administered TNF-α elicits a prolonged sympathetically mediated pressor response, but the underlying molecular mechanisms are unknown. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in cardiovascular regions of the brain has recently been recognized as a key mediator of sympathetic excitation, and ERK1/2 signaling is induced by activation of epidermal growth factor receptor (EGFR) tyrosine kinase activity. The present study examined the role of EGFR and ERK1/2 signaling in the sympathetic response to TNF-α. In urethane-anesthetized rats, intracarotid artery injection of TNF-α increased phosphorylation of EGFR and ERK1/2 in the subfornical organ (SFO) and the hypothalamic paraventricular nucleus (PVN); upregulated the gene expression of excitatory mediators in SFO and PVN; and increased blood pressure (BP), heart rate (HR), and renal sympathetic nerve activity (RSNA). A continuous intracerebroventricular infusion of the selective EGFR tyrosine kinase inhibitor AG1478 or the ERK1/2 inhibitor PD98059 significantly attenuated these responses. Bilateral PVN microinjections of TNF-α also increased phosphorylated ERK1/2 and the gene expression of excitatory mediators in PVN, along with increases in BP, HR, and RSNA, and these responses were substantially reduced by prior bilateral PVN microinjections of AG1478. These results identify activation of EGFR in cardiovascular regulatory regions of the forebrain as an important molecular mediator of TNF-α-driven sympatho-excitatory responses and suggest that EGFR activation of the ERK1/2 signaling pathway plays an essential role. These mechanisms likely contribute to sympathetic excitation in pathophysiological states like heart failure and hypertension, in which circulating and brain TNF-α levels are increased.NEW & NOTEWORTHY Proinflammatory cytokines contribute to the augmented sympathetic nerve activity in hypertension and heart failure, but the central mechanisms involved are largely unknown. The present study reveals that TNF-α transactivates EGFR in the subfornical organ and the hypothalamic paraventricular nucleus to initiate ERK1/2 signaling, upregulate the gene expression of excitatory mediators, and increase sympathetic nerve activity. These findings identify EGFR as a gateway to sympathetic excitation and a potential target for intervention in cardiovascular disease states.

Induction of Fos expression in the rat brain after intragastric administration of dried bonito dashi.

Objectives: Dried bonito dashi, a traditional Japanese fish broth made from dried bonito tuna, enhances food palatability due to its specific umami flavor characteristics. However, the pattern of brain activation following dashi ingestion has not been previously investigated.Methods: We mapped activation sites of the rat brain after intragastric loads of dried bonito dashi by measuring neuronal levels of the Fos protein, a functional marker of neuronal activation.Results: Compared to intragastric saline, intragastric dashi administration produced enhanced Fos expression in four forebrain regions: the medial preoptic area, subfornical organ, habenular nucleus, and central nucleus of the amygdala. Interestingly, the medial preoptic area was found to be the only feeding-related hypothalamic area responsive to dashi administration. Moreover, dashi had no effect in the nucleus accumbens and ventral tegmental area, two connected sites known to be activated by highly palatable sugars and fats. In the hindbrain, dashi administration produced enhanced Fos expression in both visceral sensory (caudal nucleus of the solitary tract, dorsal part of the lateral parabrachial nucleus, and area postrema) and autonomic (rostral ventrolateral medulla, and caudal ventrolateral medulla) sites.Discussion: The results demonstrate the activation of discrete forebrain and hindbrain regions following intragastric loads of dried bonito dashi. Our data suggest that the gut-brain axis is the principal mediator of the postingestive effects associated with the ingestion of dashi.

Involvement of glutamatergic mechanisms in the median preoptic nucleus in the dipsogenic response induced by angiotensinergic activation of the subfornical organ in rats.

Experiments were done to investigate the role of glutamatergic systems in the median preoptic nucleus (MnPO) in the water ingestion induced by administration of angiotensin II (ANG II) in the subfornical organ (SFO) in the awake rat. Microdialysis methods were utilized to quantify the extracellular content of glutamate (Glu) in the region of MnPO. Microinjection of ANG II (10-10 M) into the SFO significantly increased the release of Glu in the MnPO in the rats under the condition that water is available for drinking and the rats under the condition that water is not available for drinking. The amount of initial maximal increases in the Glu levels elicited by the ANG II injection was quite similar in drinking and non-drinking rats, whereas the duration of the response was much longer in non-drinking than in drinking rats. The amount of water ingestion in 20 min immediately after the ANG II injection was significantly enhanced by previous injections of N-methyl-D-aspartate (NMDA, 10 µM) into the MnPO, while the ANG II-induced water ingestion was attenuated by pretreatment with the NMDA antagonist dizocilpine (MK-801, 10 µM). The amount of water intake elicited by the ANG II injection into the SFO was enhanced by previous injections of either the non-NMDA agonist kainic acid (KA, 50 µM) or quisqualic acid (QA, 50 µM) into the MnPO. On the contrary, the ANG II-induced drinking response was diminished by pretreatment with the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM) in the MnPO. Each injection of NMDA, KA, and QA into the MnPO produced drinking behavior. These results imply that the glutamatergic neural pathways to the MnPO may transmit the information for eliciting drinking in response to ANG II acting at the SFO. Our data further provide evidence that the ANG II-induced dipsogenic response may be mediated through both NMDA and non-NMDA glutamatergic receptor mechanisms in the MnPO.

A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain.

The renin-angiotensin system (RAS) of the brain produces a series of biologically active angiotensinogen-derived peptides involved in physiological homeostasis and pathophysiology of disease. Despite significant research efforts to date, a comprehensive understanding of brain RAS physiology is lacking. A significant challenge has been the limited set of bioanalytical assays capable of detecting angiotensin (Ang) peptides at physiologically low concentrations (2-15 fmol/g of wet tissue) and sufficient chemical specificity for unambiguous molecular identifications. Additionally, a complex brain anatomy calls for microanalysis of specific tissue regions, thus further taxing sensitivity requirements for identification and quantification in studies of the RAS. To fill this technology gap, we here developed a microanalytical assay by coupling a laboratory-built capillary electrophoresis (CE) nano-electrospray ionization (nano-ESI) platform to a high-resolution mass spectrometer (HRMS). Using parallel reaction monitoring, we demonstrated that this technology achieved confident identification and quantification of the Ang peptides at approx. 5 amol to 300 zmol sensitivity. This microanalytical assay revealed differential Ang peptide profiles between tissues that were micro-sampled from the subfornical organ and the paraventricular nucleus of the hypothalamus, important brain regions involved in thirst and water homeostasis and neuroendocrine regulation to stress. Microanalytical CE-nano-ESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

Autonomic dysfunction following traumatic brain injury: translational insights.

Although there is a substantial amount of research on the neurological consequences of traumatic brain injury (TBI), there is a knowledge gap regarding the relationship between TBI and the pathophysiology of organ system dysfunction and autonomic dysregulation. In particular, the mechanisms or incidences of renal or cardiac complications after TBI are mostly unknown. Autonomic dysfunction following TBI exacerbates secondary injury and may contribute to nonneurologial complications that prolong hospital length of stay. Gaining insights into the mechanisms of autonomic dysfunction can guide advancements in monitoring and treatment paradigms to improve acute survival and long-term prognosis of TBI patients. In this paper, the authors will review the literature on autonomic dysfunction after TBI and possible mechanisms of paroxysmal sympathetic hyperactivity. Specifically, they will discuss the link among the brain, heart, and kidneys and review data to direct future research on and interventions for TBI-induced autonomic dysfunction.

Sympathoexcitation following intermittent hypoxia in rat is mediated by circulating angiotensin II acting at the carotid body and subfornical organ.

KEY POINTS: In anaesthetized rats, acute intermittent hypoxia increases sympathetic nerve activity, sympathetic peripheral chemoreflex sensitivity and central sympathetic-respiratory coupling. Renin-angiotensin system inhibition prevents the sympathetic effects of intermittent hypoxia, with intermittent injections of angiotensin II into the systemic circulation replicating these effects. Bilateral carotid body denervation reduces the sympathetic effects of acute intermittent hypoxia and eliminates the increases in chemoreflex sensitivity and sympathetic-respiratory coupling. Pharmacological inhibition of the subfornical organ also reduces the sympathetic effects of acute intermittent hypoxia, although it has no effect on the increases in chemoreflex sensitivity and central sympathetic-respiratory coupling. Combining both interventions eliminates the sympathetic effects of both intermittent hypoxia and angiotensin II. ABSTRACT: Circulating angiotensin II (Ang II) is vital for arterial pressure elevation following intermittent hypoxia in rats, although its importance in the induction of sympathetic changes is unclear. We tested the contribution of the renin-angiotensin system to the effects of acute intermittent hypoxia (AIH) in anaesthetized and ventilated rats. There was a 33.7 ± 2.9% increase in sympathetic nerve activity (SNA), while sympathetic chemoreflex sensitivity and central sympathetic-respiratory coupling increased by one-fold following AIH. The sympathetic effects of AIH were prevented by blocking angiotensin type 1 receptors with systemic losartan. Intermittent systemic injections of Ang II (Int.Ang II) elicited similar sympathetic responses to AIH. To identify the neural pathways responsible for the effects of AIH and Int.Ang II, we performed bilateral carotid body denervation, which reduced the increase in SNA by 56% and 45%, respectively. Conversely, pharmacological inhibition of the subfornical organ (SFO), an established target of circulating Ang II, reduced the increase in SNA following AIH and Int.Ang II by 65% and 59%, respectively, although it did not prevent the sensitization of the sympathetic peripheral chemoreflex, nor the increase in central sympathetic-respiratory coupling. Combined carotid body denervation and inhibition of the SFO eliminated the enhancement of SNA following AIH and Int.Ang II. Repeated systemic injections of phenylephrine caused an elevation in SNA similar to AIH, and this effect was prevented by a renin inhibitor, aliskiren. Our findings show that the sympathetic effects of AIH are the result of RAS-mediated activations of the carotid bodies and the SFO.

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.

Increased (pro)renin receptor expression in the subfornical organ of hypertensive humans.

The central nervous system plays an important role in essential hypertension in humans and in animal models of hypertension through modulation of sympathetic activity and Na+ and body fluid homeostasis. Data from animal models of hypertension suggest that the renin-angiotensin system in the subfornical organ (SFO) of the brain is critical for hypertension development. We recently reported that the brain (pro)renin receptor (PRR) is a novel component of the brain renin-angiotensin system and could be a key initiator of the pathogenesis of hypertension. Here, we examined the expression level and cellular distribution of PRR in the SFO of postmortem human brains to assess its association with the pathogenesis of human hypertension. Postmortem SFO tissues were collected from hypertensive and normotensive human subjects. Immunolabeling for the PRR and a retrospective analysis of clinical data were performed. We found that human PRR was prominently expressed in most neurons and microglia, but not in astrocytes, in the SFO. Importantly, PRR levels in the SFO were elevated in hypertensive subjects. Moreover, PRR immunoreactivity was significantly correlated with systolic blood pressure but not body weight, age, or diastolic blood pressure. Interestingly, this correlation was independent of antihypertensive drug therapy. Our data indicate that PRR in the SFO may be a key molecular player in the pathogenesis of human hypertension and, as such, could be an important focus of efforts to understand the neurogenic origin of hypertension. NEW & NOTEWORTHY This study provides evidence that, in the subfornical organ of the human brain, the (pro)renin receptor is expressed in neurons and microglia cells but not in astrocytes. More importantly, (pro)renin receptor immunoreactivity in the subfornical organ is increased in hypertensive humans and is significantly correlated with systolic blood pressure.

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.

Vasopressin and the Regulation of Thirst.

Recent experiments using optogenetic tools allow the identification and functional analysis of thirst neurons and vasopressin producing neurons. Two major advances provide a detailed anatomy of taste for water and arginine-vasopressin (AVP) release: (1) thirst and AVP release are regulated not only by the classical homeostatic, intero-sensory plasma osmolality negative feedback, but also by novel, extero-sensory, anticipatory signals. These anticipatory signals for thirst and vasopressin release converge on the same homeostatic neurons of circumventricular organs that monitor the composition of the blood; (2) acid-sensing taste receptor cells (which express polycystic kidney disease 2-like 1 protein) on the tongue that were previously suggested as the sour taste sensors also mediate taste responses to water. The tongue has a taste for water. The median preoptic nucleus (MnPO) of the hypothalamus could integrate multiple thirst-generating stimuli including cardiopulmonary signals, osmolality, angiotensin II, oropharyngeal and gastric signals, the latter possibly representing anticipatory signals. Dehydration is aversive and MnPO neuron activity is proportional to the intensity of this aversive state.

The proinflammatory cytokine tumor necrosis factor-α excites subfornical organ neurons.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine implicated in cardiovascular and autonomic regulation via actions in the central nervous system. TNF-α-/- mice do not develop angiotensin II (ANG II)-induced hypertension, and administration of TNF-α into the bloodstream of rats increases blood pressure and sympathetic tone. Recent studies have shown that lesion of the subfornical organ (SFO) attenuates the hypertensive and autonomic effects of TNF-α, while direct administration of TNF-α into the SFO increases blood pressure, suggesting the SFO to be a key site for the actions of TNF-α. Therefore, we used patch-clamp techniques to examine both acute and long-term effects of TNF-α on the excitability of Sprague-Dawley rat SFO neurons. It was observed that acute bath application of TNF-α depolarized SFO neurons and subsequently increased action potential firing rate. Furthermore, the magnitude of depolarization and the proportion of depolarized SFO neurons were concentration dependent. Interestingly, following 24-h incubation with TNF-α, the basal firing rate of the SFO neurons was increased and the rheobase was decreased, suggesting that TNF-α elevates SFO neuron excitability. This effect was likely mediated by the transient sodium current, as TNF-α increased the magnitude of the current and lowered its threshold of activation. In contrast, TNF-α did not appear to modulate either the delayed rectifier potassium current or the transient potassium current. These data suggest that acute and long-term TNF-α exposure elevates SFO neuron activity, providing a basis for TNF-α hypertensive and sympathetic effects.NEW & NOTEWORTHY Considerable recent evidence has suggested important links between inflammation and the pathological mechanisms underlying hypertension. The present study describes cellular mechanisms through which acute and long-term exposure of tumor necrosis factor-α (TNF-α) influences the activity of subfornical organ neurons by modulating the voltage-gated transient Na+ current. This provides critical new information regarding the specific pathological mechanisms through which inflammation and TNF-α in particular may result in the development of hypertension.

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.

Subfornical organ neurons integrate cardiovascular and metabolic signals.

The subfornical organ (SFO) is a critical circumventricular organ involved in the control of cardiovascular and metabolic homeostasis. Despite the plethora of circulating signals continuously sensed by the SFO, studies investigating how these signals are integrated are lacking. In this study, we use patch-clamp techniques to investigate how the traditionally classified "cardiovascular" hormone ANG II, "metabolic" hormone CCK and "metabolic" signal glucose interact and are integrated in the SFO. Sequential bath application of CCK (10 nM) and ANG (10 nM) onto dissociated SFO neurons revealed that 63% of responsive SFO neurons depolarized to both CCK and ANG; 25% depolarized to ANG only; and 12% hyperpolarized to CCK only. We next investigated the effects of glucose by incubating and recording neurons in either hypoglycemic, normoglycemic, or hyperglycemic conditions and comparing the proportions of responses to ANG (n = 55) or CCK (n = 83) application in each condition. A hyperglycemic environment was associated with a larger proportion of depolarizing responses to ANG (χ2, P < 0.05), and a smaller proportion of depolarizing responses along with a larger proportion of hyperpolarizing responses to CCK (χ2, P < 0.01). Our data demonstrate that SFO neurons excited by CCK are also excited by ANG and that glucose environment affects the responsiveness of neurons to both of these hormones, highlighting the ability of SFO neurons to integrate multiple metabolic and cardiovascular signals. These findings have important implications for this structure's role in the control of various autonomic functions during hyperglycemia.