Mechanisms of GABA-mediated inhibition of the angiotensin II-induced cytosolic Ca2+ increase in rat subfornical organ neurons.

Neurons in the subfornical organ (SFO) sense both neurotransmitters and circulating humoral factors such as angiotensin II (AII) and atrial natriuretic peptide (ANP), and regulate multiple physiological functions including drinking behavior. We recently reported that AII at nanomolar concentrations induced a persistent [Ca2+]i increase in acutely dissociated SFO neurons and that this effect of AII was reversibly inhibited by GABA. In the present study, we studied the inhibitory mechanism of GABA using Ca2+ imaging and patch-clamp electrophysiology. The AII-induced persistent [Ca2+]i increase was inhibited by GABA in more than 90% of AII-responsive neurons and by other two SFO inhibitory ligands, ANP and galanin, in about 60 and 30% of neurons respectively. The inhibition by GABA was mimicked by the GABAA and GABAB receptor agonists muscimol and baclofen. The involvement of both GABA receptor subtypes was confirmed by reversal of the GABA-mediated inhibition only when the GABAA and GABAB receptors antagonists bicuculline methiodide and CGP55845 were both present. The GABAB agonist baclofen rapidly and reversibly inhibited voltage-gated Ca2+ channel (VGCC) currents recorded in response to depolarizing pulses in voltage-clamp electrophysiology using Ba2+ as a charge carrier (IBa). Baclofen inhibition of IBa was antagonized by CGP55845, confirming GABAB receptor involvement; was reduced by N-ethylmaleimide, suggesting downstream Gi-mediated actions; and was partially removed by a large prepulse, indicating voltage-dependency. The magnitude of IBa inhibition by baclofen was reduced by the application of selective blockers for N-, P/Q-, and L-type VGCCs (ω-conotoxin GVIA, ω-agatoxin IVA, and nifedipine respectively). Overall, our study indicates that GABA inhibition of the AII-induced [Ca2+]i increase is mediated by both GABAA and GABAB receptors, and that GABAB receptors associated with Gi proteins suppress Ca2+ entry through VGCCs in SFO neurons.

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.

Redox-sensitive calcium/calmodulin-dependent protein kinase IIα in angiotensin II intra-neuronal signaling and hypertension.

Dysregulation of brain angiotensin II (AngII) signaling results in modulation of neuronal ion channel activity, an increase in neuronal firing, enhanced sympathoexcitation, and subsequently elevated blood pressure. Studies over the past two decades have shown that these AngII responses are mediated, in part, by reactive oxygen species (ROS). However, the redox-sensitive target(s) that are directly acted upon by these ROS to execute the AngII pathophysiological responses in neurons remain unclear. Calcium/calmodulin-dependent protein kinase II (CaMKII) is an AngII-activated intra-neuronal signaling protein, which has been suggested to be redox sensitive as overexpressing the antioxidant enzyme superoxide dismutase attenuates AngII-induced activation of CaMKII. Herein, we hypothesized that the neuronal isoform of CaMKII, CaMKII-alpha (CaMKIIα), is a redox-sensitive target of AngII, and that mutation of potentially redox-sensitive amino acids in CaMKIIα influences AngII-mediated intra-neuronal signaling and hypertension. Adenoviral vectors expressing wild-type mouse CaMKIIα (Ad.wtCaMKIIα) or mutant CaMKIIα (Ad.mutCaMKIIα) with C280A and M281V mutations were generated to overexpress either CaMKIIα isoform in mouse catecholaminergic cultured neurons (CATH.a) or in the brain subfornical organ (SFO) of hypertensive mice. Overexpressing wtCaMKIIα exacerbated AngII pathophysiological responses as observed by a potentiation of AngII-induced inhibition of voltage-gated K+ current, enhanced in vivo pressor response following intracerebroventricular injection of AngII, and sensitization to chronic peripheral infusion of AngII resulting in a more rapid increase in blood pressure. In contrast, expressing the mutant CaMKIIα in CATH.a neurons or the SFO failed to intensify these AngII responses. Taken together, these data identify neuronal CaMKIIα as a redox-sensitive signaling protein that contributes to AngII-induced neuronal activation and hypertension.

Persistent cytosolic Ca2+ increase induced by angiotensin II at nanomolar concentrations in acutely dissociated subfornical organ (SFO) neurons of rats.

It is known that angiotensin II (AII) is sensed by subfornical organ (SFO) to induce drinking behaviors and autonomic changes. AII at picomolar concentrations have been shown to induce Ca2+ oscillations and increase in the amplitude and frequency of spontaneous Ca2+ oscillations in SFO neurons. The present study was conducted to examine effects of nanomolar concentrations of AII using the Fura-2 Ca2+-imaging technique in acutely dissociated SFO neurons. AII at nanomolar concentrations induced an initial [Ca2+]i peak followed by a persistent [Ca2+]i increase lasting for longer than 1 hour. By contrast, [Ca2+]i responses to 50 mM K+, maximally effective concentrations of glutamate, carbachol, and vasopressin, and AII given at picomolar concentrations returned to the basal level within 20 min. The AII-induced [Ca2+]i increase was blocked by the AT1 antagonist losartan. However, losartan had no effect when added during the persistent phase. The persistent phase was suppressed by extracellular Ca2+ removal, significantly inhibited by blockers of L and P/Q type Ca2+ channels , but unaffected by inhibition of Ca2+ store Ca2+ ATPase. The persistent phase was reversibly suppressed by GABA and inhibited by CaMK and PKC inhibitors. These results suggest that the persistent [Ca2+]i increase evoked by nanomolar concentrations of AII is initiated by AT1 receptor activation and maintained by Ca2+ entry mechanisms in part through L and P/Q type Ca2+ channels, and that CaMK and PKC are involved in this process. The persistent [Ca2+]i increase induced by AII at high pathophysiological levels may have a significant role in altering SFO neuronal functions.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

AT1 receptors in the subfornical organ modulate arterial pressure and the baroreflex in two-kidney, one-clip hypertensive rats.

The subfornical organ (SFO), a forebrain circumventricular organ that lies outside the blood-brain barrier, has been implicated in arterial pressure and baroreflex responses to angiotensin II (ANG II). We tested whether pharmacological inhibition or selective silencing of SFO ANG II type 1 receptors (AT1R) of two-kidney, one-clip rats with elevated plasma ANG II decreases resting arterial pressure and renal sympathetic nerve activity (RSNA) and/or modulates arterial baroreflex responses of heart rate (HR) and RSNA. Male Sprague-Dawley rats underwent renal artery clipping [2-kidney, 1-clip (2K,1C)] or sham clipping (sham). After 6 wk, conscious rats instrumented with vascular catheters, renal nerve electrodes, and a cannula directed to the SFO were studied. In another set of experiments, rats were instrumented with hemodynamic and nerve radio transmitters and injected with scrambled RNA or silencing RNA targeted against AT1R. Mean arterial pressure (MAP) was significantly higher in 2K,1C rats. Acute SFO injection with the AT1R inhibitor losartan did not change MAP in sham or 2K,1C rats. Baroreflex curves of HR and RSNA were shifted rightward in 2K,1C rats. Losartan exerted no effect. SFO AT1R knockdown did not influence MAP in sham rats but decreased MAP in 2K,1C rats, despite no change in plasma ANG II or resting RSNA. AT1R knockdown prevented the reduction in maximum gain and slope of baroreflex responses of HR and RSNA; the reduced RSNA response to baroreceptor unloading was partially restored in 2K,1C rats. These findings show that AT1R activation within the SFO contributes to hypertension and baroreflex dysfunction in 2K,1C rats and highlight the temporal requirement for reversal of these effects.

The cytosolic Ca2+ concentration in acutely dissociated subfornical organ (SFO) neurons of rats: Spontaneous Ca2+ oscillations and Ca2+ oscillations induced by picomolar concentrations of angiotensin II.

Characteristics of subfornical organ (SFO) neurons were examined by measuring the cytosolic Ca2+ concentration ([Ca2+]i) in acutely dissociated neurons of the rat. SFO neurons, defined by the responsiveness to 50 mM K+ (n = 67) responded to glutamate (86%), angiotensin II (AII) (50%), arginine vasopressin (AVP) (66%) and/or carbachol (CCh) (61%), at their maximal concentrations, with marked increases in [Ca2+]i. More than a half (174/307) of SFO neurons examined exhibited spontaneous Ca2+ oscillations, while the remainder showed a relatively stable baseline under unstimulated conditions. Spontaneous Ca2+ oscillations were suppressed when extracellular Ca2+ was removed and were inhibited when extracellular Na+ was replaced with equimolar N-methyl-D-glucamine. Ca2+ oscillations were unaffected by the inhibitor of Ca2+-dependent ATPases cyclopiazonic acid, the N-type Ca2+ channel blocker ω-conotoxin GVIA and the P/Q-type Ca2+ channel blocker ω-agatoxin IVA, but significantly inhibited by the high-voltage-activated Ca2+ channel blocker Cd2+ and the L-type Ca2+ channel blocker nicardipine. Ca2+ oscillations were also completely arrested by the voltage-gated Na+ channel blocker tetrodotoxin in 50% of SFO neurons but only partially in the remaining neurons. These results suggest that SFO neurons exhibit spontaneous membrane Ca2+ oscillations that are dependent in part on Ca2+ entry through L-type Ca2+ channels, whose activation may result from burst firing. Moreover, AII at picomolar concentrations induced Ca2+ oscillations in neurons showing no spontaneous Ca2+ oscillations, while spontaneous Ca2+ oscillations were arrested by gamma-aminobutyric acid (10 µM), suggesting that rises in [Ca2+]i during Ca2+ oscillations may play an important role in the modulation of SFO neuron function.

The subfornical organ in sodium appetite: Recent insights.

To maintain sodium homeostasis, animals will readily seek and ingest salt when salt-depleted, even at concentrations that they typically find aversive when sodium replete. This innate behaviour is known as sodium (or salt) appetite. Salt appetite is subserved by a conserved brain network that senses sodium need and promotes the ingestion of salty substances when sodium-deficient. The subfornical organ (SFO) is a circumventricular organ that has diverse roles encompassing cardiovascular regulation, energy balance, immune responses, reproduction, and hydromineral balance. The SFO acts as a central sensor of sodium need and is essential for the generation of salt appetite. In this review, we discuss recent findings on the neurochemical and circuit-level organisation of the SFO in the context of sodium appetite. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Interaction between angiotensin II and glucose sensing at the subfornical organ.

The subfornical organ (SFO) lacks the normal blood-brain barrier and senses the concentrations of many different circulating signals, including glucose and angiotensin II (ANG II). ANG II has recently been implicated in the control of food intake and body weight gain. The present study assessed whether single SFO neurones sense changes in glucose and ANG II, and also whether changes in glucose concentration alter the responsiveness of these neurones to ANG II. SFO neurones dissociated from male Sprague-Dawley rats (100-175 g) were used. We first examined whether glucose concentration modulates AT1 receptor expression. Similar AT1a mRNA expression levels were found at glucose concentrations of 1, 5 and 10 mmol L-1 in dissociated SFO neurones. Glucose responsiveness of SFO neurones was assessed using perforated current-clamp recordings and switching between 5 and 10 mmol L-1 glucose artificial cerebrospinal fluid to classify single neurones as nonresponsive (nGS), glucose-excited (GE) or glucose-inhibited (GI). In total, 26.7% of the SFO neurones were GI (n = 24 of 90), 21.1% were GE (n = 19 of 90) and 52.2% were nGS (n = 47 of 90). Once classified, the effects of 10 nmol L-1 ANG II on the excitability of these neurones were tested, with 52% of GE (n = 10 of 19), 71% of GI (n = 17 of 24) and 43% of nGS (n = 20 of 47) neurones being ANG II sensitive. Finally, we tested whether acute changes in glucose concentration modified the response to ANG II and showed that some neurones (4/17) only respond to ANG II at 10 mmol L-1 glucose. Our data demonstrate that the same SFO neurone can sense glucose and ANG II and that acute changes in glucose concentration may change ANG II responsiveness.

The subfornical organ: A novel site for prolactin action.

Prolactin (PRL) is a peptide hormone that performs over 300 biological functions, including those that require binding to prolactin receptor (PRL-R) in neurones within the central nervous system (CNS). To enter the CNS, circulating PRL must overcome the blood-brain barrier. Accordingly, areas of the brain that do not possess a blood-brain barrier, such as the subfornical organ (SFO), are optimally positioned to interact with systemic PRL. The SFO has been classically implicated in energy and fluid homeostasis but has the potential to influence oestrous cyclicity and gonadotrophin release, which are also functions of PRL. We aimed to confirm and characterise the expression of PRL-R in the SFO, as well as identify the effects of PRL application on membrane excitability of dissociated SFO neurones. Using a quantitative real-time polymerase chain reaction, we found that PRL-R mRNA in the SFO of male and female Sprague Dawley rats did not significantly differ between juvenile and sexually mature rats (P = .34), male and female rats (P = .97) or across the oestrous cycle (P = .54). Patch-clamp recordings were obtained in juvenile male rats to further investigate the actions of PRL at the SFO. Dissociated SFO neurones perfused with 1 µmol L-1 PRL resulted in 2 responsive subpopulations of neurones; 40% depolarised (n = 15/43, 11.3 ± 1.7 mV) and 14% hyperpolarised (n = 6/43, -6.7 ± 1.4 mV) to PRL application. Within the range of 10 pmol L-1 to 1 µmol L-1 , the concentrations of PRL were not significantly different in either the magnitude (P = .53) or proportion (P = .19) of response. Furthermore, PRL application significantly reduced the transient K+ current in 67% of SFO neurones in voltage-clamp configuration (n = 6/9, P = .02). The stability in response to PRL and expression of PRL-R in the SFO suggests that PRL function is conserved across physiological states and circulating PRL concentrations, prompting further investigations aiming to clarify the nature of PRL function in the SFO.

Brain-derived neurotrophic factor acts at neurons of the subfornical organ to influence cardiovascular function.

Brain-derived neurotrophic factor (BDNF), a neurotrophin traditionally associated with neural plasticity, has more recently been implicated in fluid balance and cardiovascular regulation. It is abundantly expressed in both the central nervous system (CNS) and peripheral tissue, and is also found in circulation. Studies suggest that circulating BDNF may influence the CNS through actions at the subfornical organ (SFO), a circumventricular organ (CVO) characterized by the lack of a normal blood-brain barrier (BBB). The SFO, well-known for its involvement in cardiovascular regulation, has been shown to express BDNF mRNA and mRNA for the TrkB receptor at which BDNF preferentially binds. This study was undertaken to determine if: (1) BDNF influences the excitability of SFO neurons in vitro; and (2) the cardiovascular consequences of direct administration of BDNF into the SFO of anesthetized rats. Electrophysiological studies revealed that bath application of BDNF (1 nmol/L) influenced the excitability of the majority of neurons (60%, n = 13/22), the majority of which exhibited a membrane depolarization (13.8 ± 2.5 mV, n = 9) with the remaining affected cells exhibiting hyperpolarizations (-11.1 ± 2.3 mV, n = 4). BDNF microinjections into the SFO of anesthetized rats caused a significant decrease in blood pressure (mean [area under the curve] AUC = -364.4 ± 89.0 mmHg × sec, n = 5) with no effects on heart rate (mean AUC = -12.2 ± 3.4, n = 5). Together these observations suggest the SFO to be a CNS site at which circulating BDNF could exert its effects on cardiovascular regulation.

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.

Blood-borne interleukin-1ß acts on the subfornical organ to upregulate the sympathoexcitatory milieu of the hypothalamic paraventricular nucleus.

We previously reported that microinjection of the proinflammatory cytokine interleukin-1ß (IL-1ß) into the subfornical organ (SFO) elicits a pressor response accompanied by increases in inflammation and renin-angiotensin system (RAS) activity in the SFO and hypothalamic paraventricular nucleus (PVN). The present study sought to determine whether blood-borne IL-1ß induces similar neurochemical changes in the SFO and PVN and, if so, whether increased inflammation and RAS activity at the SFO level orchestrate the sympathoexcitatory response to circulating IL-1ß. In urethane-anesthetized male Sprague-Dawley rats, intravenous injection of IL-1ß (500 ng) increased blood pressure, heart rate, renal sympathetic nerve activity, and mRNA for angiotensin-converting enzyme, angiotensin II type 1a receptor, cyclooxygenase-2, tumor necrosis factor-α, and IL-1ß, as well as the tumor necrosis factor-α p55 receptor and the IL-1 receptor, in the SFO and PVN. Pretreatment with SFO microinjections of the angiotensin II type 1a receptor blocker losartan (1 µg), the angiotensin-converting enzyme inhibitor captopril (1 µg), or the cyclooxygenase-2 inhibitor NS-398 (2 µg) attenuated expression of these excitatory mediators in the SFO and downstream in the PVN and the IL-1ß-induced pressor responses. An SFO lesion minimized the IL-1ß-induced expression of inflammatory and RAS components as well as c-Fos, an indicator of neuronal excitation, in the PVN. These studies demonstrate that circulating IL-1ß, which increases in cardiovascular disorders such as hypertension and heart failure, acts on the SFO to increase inflammation and RAS activity in the SFO and PVN and that intervening in these neurochemical processes in the SFO can significantly reduce the sympathetic response.

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.

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.

Noradrenaline receptor mechanisms modulate the angiotensin II-induced water intake in the subfornical organ in rats.

The present study was carried out to clarify the role of noradrenergic systems in the mediation of drinking response to angiotensin II (ANG II) in the rat subfornical organ (SFO). Microinjection of ANG II (10 pmol, 50 nl) into the SFO caused a robust drinking response (water volume, 1.8-8.7 ml for 20 min). Injections of either noradrenaline (NA; 0.1, 1 and 10 nmols, 50 nl) into the SFO did not produce a significant water intake. Phenylephrine (Phen; 0.1, 1, 10 nmols, 50 nl), an α1-adrenoceptor agonist, injected into the SFO elicited little drinking in the rats tested (water volume, 0.4-1.5 ml for 20 min). Previous injections of NA (0.1 and 1 nmols) or Phen (0.1, 1 and 10 nmols) significantly enhanced the water intake elicited by the injection of ANG II into the SFO. Neither the α2-adrenoceptor agonist clonidine (Clon; 10 nmol, 50 nl) nor the ß-adrenoceptor agonist isoprenaline (Isop; 10 nmol, 50 nl) into the SFO caused a significant water intake. Previous injections of Clon (0.1, 1 and 10 nmol, 50 nl) into the SFO were without effect on the water intake produced by the ANG II injection into the SFO. Pretreatment with Isop (1 and 10 nmols), on the other hand, significantly attenuated the drinking response to ANG II. Vehicle (artificial cerebrospinal fluid, 50 nl) had no effect on the ANG II-induced water intake. These results suggest that both α1-(facilitatory) and ß-(inhibitory) adrenoceptor mechanisms may be implicated in the control of drinking response induced by angiotensinergic activation of SFO neurons.

Changes in endothelial cell proliferation and vascular permeability after systemic lipopolysaccharide administration in the subfornical organ.

The subfornical organ (SFO) has highly permeable fenestrated vasculature and is a key site for immune-to-brain communications. Recently, we showed the occurrence of continuous angiogenesis in the SFO. In the present study, we found that systemic administration of bacterial lipopolysaccharide (LPS) reduced the vascular permeability and endothelial cell proliferation. In LPS-administered mice, the SFO vasculature showed a significant decrease in the immunoreactivity of plasmalemma vesicle associated protein-1, a marker of endothelial fenestral diaphragms. These data suggest that vasculature undergoes structural change to decrease vascular permeability in response to systemic LPS administration.

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

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

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.

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.