Rat Subfornical Organ Research Articles

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.

Catecholaminergic Structures of the Rat Subfornical Organ

Catecholaminergic Structures of the Rat Subfornical Organ

Микроглия и предполагаемые макрофаги субфорникального органа: структурно-функциональные особенности

The subfornical organ is an important regulator of water-salt metabolism and energy balance of the body, involved in the control of the cardiovascular system and immune regulation. The organ comprises several cell populations, among which microglia and macrophages remain uncharacterized. This study aimed at structural, cytochemical, and functional characterization of microglia and macrophages of the subfornical organ in rats. Brain specimens were collected from mature male Wistar rats (n = 8). Microglia and macrophages were revealed by immunostaining with poly- and monoclonal antibodies against calcium-binding protein Iba1 and lysosomal protein CD68; the slides were examined by light and confocal laser microscopy. The study provides a comprehensive morphological characterization of microglial cells and macrophages of the subfornical organ. We demonstrate that the majority of Iba1-expressing cells in this area of the brain are microglial cells, not macrophages. Pre-activated state of the subfornical organ microglia may reflect structural and functional features of this organ and specific functions of local microglia. Subependymal microglial cells, the processes of which penetrate into the third ventricle of the brain, constitute a distinct subpopulation among the Iba1-expressing cells of the subfornical organ. Apart from microglial elements, the subfornical organ contains few tissue macrophages with characteristic strong expression of CD68 accompanied by undetectable or weak expression of Iba1.

Anatomical Organization of the Rat Subfornical Organ.

The subfornical organ (SFO) is a sensory circumventricular organ located along the anterodorsal wall of the third ventricle. SFO lacks a complete blood-brain barrier (BBB), and thus peripherally-circulating factors can penetrate the SFO parenchyma. These signals are detected by local neurons providing the brain with information from the periphery to mediate central responses to humoral signals and physiological stressors. Circumventricular organs are characterized by the presence of unique populations of non-neuronal cells, such as tanycytes and fenestrated endothelium. However, how these populations are organized within the SFO is not well understood. In this study, we used histological techniques to analyze the anatomical organization of the rat SFO and examined the distribution of neurons, fenestrated and non-fenestrated vasculature, tanycytes, ependymocytes, glia cells, and pericytes within its confines. Our data show that the shell of SFO contains non-fenestrated vasculature, while fenestrated capillaries are restricted to the medial-posterior core region of the SFO and associated with a higher BBB permeability. In contrast to non-fenestrated vessels, fenestrated capillaries are encased in a scaffold created by pericytes and embedded in a network of tanycytic processes. Analysis of c-Fos expression following systemic injections of angiotensin II or hypertonic NaCl reveals distinct neuronal populations responding to these stimuli. Hypertonic NaCl activates ∼13% of SFO neurons located in the shell. Angiotensin II-sensitive neurons represent ∼35% of SFO neurons and their location varies between sexes. Our study provides a comprehensive description of the organization of diverse cellular elements within the SFO, facilitating future investigations in this important brain area.

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 (10-10M) 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 20min immediately after the ANG II injection was significantly enhanced by previous injections of N-methyl-D-aspartate (NMDA, 10μM) into the MnPO, while the ANG II-induced water ingestion was attenuated by pretreatment with the NMDA antagonist dizocilpine (MK-801, 10μM). 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, 50μM) or quisqualic acid (QA, 50μM) 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, 10μM) 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.

Abstract P290: Central Angiotensin II and Interleukin-1©¬ Upregulate Brain Tumor Necrosis Factor-¥å Converting Enzyme in the Rat

Tumor necrosis factor-α (TNF-α) converting enzyme (TACE/ADAM17) proteolytically cleaves the extracellular domain of transmembrane TNF-α to release the soluble form of TNF-α that contributes to augmented sympathetic nerve activity in heart failure (HF) and hypertension (HTN). Our previous study indicated that TACE is upregulated in the subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN), two key cardiovascular and autonomic brain centers, in rats with ischemia-induced HF, and is associated with TNF-α –induced sympathetic excitation in that setting. However, the mechanisms regulating TACE activity in the brain remain a mystery. The present study sought to determine whether angiotensin II (ANG II) or interleukin-1β (IL-1β), which increase in SFO and PVN in HF, upregulate TACE expression. Urethane anesthetized male SD rats underwent a 4- hour intracerebroventricular (ICV) infusion of artificial cerebrospinal fluid (aCSF), ANG II (100 ng/hr) or IL-1β (50 ng/hr). Some rats were euthanized to collect SFO and PVN tissues to measure TACE mRNA and protein; others were transcardially perfused with 4% paraformaldehyde to immunostain for TACE in SFO and PVN. ICV infusion of ANG II (n=6) or IL-1β (n=6) significantly (*p<0.05) increased TACE mRNA level (fold change) in SFO (2.54 ± 0.24* and 2.36 ± 0.21*, respectively) and PVN (2.39* ± 0.25 and 2.23 ± 0.20*, respectively) compared with ICV aCSF infusion. Western blot analysis also revealed a significant increase in the TACE protein (normalized to β-actin) in ANG II or IL-1β infused rats in SFO (0.31±0.06* and 0.26±0.05* respectively) and PVN (0.27 ± 0.04*, 0.24 ± 0.03*, respectively) compared with ICV aCSF-infused rats (SFO: 0.12±0.05; PVN: 0.09±0.04). Confocal images exhibited that TACE-like immunoreactivity was markedly higher in all four subdivisions of PVN and in SFO in rats treated with ICV ANG II or IL-1β, compared with aCSF-infused rats. These data indicate that ANG II and IL-1β upregulate TACE expression in SFO and PVN, suggesting that activation of the renin-angiotensin system and augmented inflammation may upregulate TACE expression in the SFO and PVN in HF and HTN. TACE-mediated ectodomain shedding in SFO and PVN may contribute to ANG II- and cytokine-driven sympathoexcitation in HF and HTN.

The transcriptome of the rat subfornical organ is altered in response to early postnatal overnutrition

Early postnatal overnutrition in humans is associated with long-term negative outcomes including obesity, increased risk of type-II diabetes, and cardiovascular disease. Hypothalamic neurons from rodents exposed to early postnatal overnutrition show altered expression of satiety signals and receptors, and exhibit altered responses to many satiety signals, suggesting a hypothalamic link between early overnutrition and development of these sequelae. Importantly, several hypothalamic nuclei receive information regarding circulating hormones (such as insulin, leptin and ghrelin) from the subfornical organ (SFO), a forebrain sensory circumventricular organ which lacks a blood brain barrier. Previous transcriptomic studies indicate that challenges to energy balance and hydration status stimulate changes in gene expression within the SFO, including genes encoding ion channels and receptors. In order to determine if early postnatal overnutrition also causes changes in SFO gene expression which may be associated with homeostatic dysregulation, we performed whole transcriptome sequencing on SFO tissue from rats raised in small (4 pups), or control (large, 12 pups) litters. Illumina RNA sequencing was performed on SFO tissue from rats raised from small and large litters, and read sequences were aligned to the Rat Rnor_6.0 genome. Control data were further compared to previously published microarray data set for validation. We found statistically significant (p<0.05) changes in expression of 12 transcripts, three of which have likely roles in neuronal excitability, neurite outgrowth and differentiation, and food intake (Manf, Slc24a4, Cracr2b). Additionally, gene ontology analysis identified a trend among significantly altered transcripts in roles for oxidative stress response. We conclude that the SFO transcriptome is subtly altered by early postnatal overnutrition, and recommend further investigation of the effect of early postnatal overnutrition on SFO physiology and morphology.

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

GABAergic modulation of serotonin release in the rat subfornical organ area

The present study was carried out to examine whether γ–aminobutyric acid (GABA) receptor mechanisms are involved in the release of serotonin (5-hydroxytryptamine, 5-HT) in the subfornical organ (SFO) using intracerebral microdialysis techniques. Perfusion with the GABA receptor antagonists as well as agonists was performed in the region of the SFO through a microdialysis probe and extracellular concentrations of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in freely moving rats. Perfusion with the GABAA receptor antagonist bicuculline (10 and 50μM), but not the GABAB receptor antagonist phaclofen (10 and 50μM), increased dialysate 5-HT and 5-HIAA concentrations in the SFO area, suggesting that the GABAergic system may tonically inhibit the 5-HT release in the SFO area through GABAA receptors. Higher perfusion with the GABAA receptor agonist muscimol (50μM) or the GABAB receptor agonist baclofen (250μM) decreased extracellular levels of 5-HT and 5-HIAA in the SFO area. Nonhypotensive hypovolemia induced by subcutaneous injection of polyethylene glycol (PEG, 30%, 5ml) significantly enhanced the 5-HT and 5-HIAA concentrations in the SFO area. The enhanced 5-HT and 5-HIAA levels elicited the PEG treatment were reduced by perfusion with muscimol (10μM), but not by baclofen (50μM). These results show the involvement of both GABAA and GABAB receptors in the modulation of the 5-HT release in the SFO area, and imply that the GABAA receptor mechanism may be importance for the serotonergic regulatory system of body fluid balance.

Suppression of TRPV4 channels ameliorates anti-dipsogenic effects under hypoxia in the subfornical organ of rats.

The phenomenon of water intake reduction during the 1st day of hypobaric hypoxia has been known for a long time. However, the reason for the same is yet unknown. The transient receptor potential vanilloid (TRPV) channels, including TRPV1 and TRPV4, are located in the subfornical organ (SFO). These are calcium permeable cationic channels gated by various stimuli such as cell swelling, low pH, and high temperature, and participate in anti-dipsogenic effects when activated. We aimed to explore the drinking behavior of rats and the mechanism of TRPVs under hypoxia. Chemical TRPV4 inhibitors (HC-067047 and Gadolinium) or TRPV4 knockout, but not TRPV1 inhibitor SB-705498, could restore the water intake under hypoxia. Hypoxia-mediated direct activation of TRPV4 may be the reason of anti-dipsogenic effects because the serum sodium, pH, and intracranial temperature are unaltered. Interestingly, we found that hypoxia immediately increased the intracellular Ca2+ concentration ([Ca2+]i) in HEK293-TRPV4 cells and primary neurons from SFO region, but not in the HEK293-TRPV1 cells. Moreover, hypoxia-induced [Ca2+]i increase depended on the indispensable hemeoxygenase-2 (HO-2) and TRPV4. HO-2 and TRPV4 were also confirmed to form a complex in SFO neurons. These results demonstrated that SFO cells sense hypoxia and activate via the HO-2/TRPV4 multiple channels, which are associated with anti-dipsogenic effects.

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.

Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups

The transcription factor SOX2 has many established roles in neural development but is generally considered to have limited activity in the adult brain. As part of a study of neuronal phenotypes in the adult rodent hypothalamus, we have now used immunohistochemical analysis to investigate the expression of SOX2 in the adult rat and mouse hypothalamus. Our analysis has revealed that SOX2 protein is extensively expressed in cells of the suprachiasmatic nucleus (SCN). Co-localization with the nuclear marker proteins NeuN and MeCP2 confirmed SOX2 expression in mature neurons of the rat SCN, and the functional integrity of these SOX2+ neurons was also confirmed by demonstrating co-localization with light-induced EGR1 protein. In addition to the SCN, we have also revealed a population of SOX2+/(NeuN+/MeCP2+) neurons in the rat periventricular nucleus (PeN). However, in other hypothalamic nuclei such as the supraoptic nucleus (SON) SOX2+ cells were rare. In extra-hypothalamic areas, SOX2+ cells were also scarce although we have confirmed populations of non-neuronal SOX2+ cells in both the rat sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the hippocampus. In addition, we have identified an extensive, novel population of non-neuronal SOX2+ cells in the rat subfornical organ (SFO). Our findings provide further evidence of ‘immature’ phenotypes in rodent SCN neurons and, given the extensive expression of SOX2 across these hypothalamic neurons, may identify a common regulatory factor that maintains this unusual neuronal phenotype. Conservation of SCN SOX2 expression in both rat and mouse indicates a functional requirement for this transcription factor that may be integral to the role of these SCN neurons in mediating daily physiological rhythms.

ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats.

Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure.

Effects of exercise training on SFO-mediated sympathoexcitation during chronic heart failure

Exercise training (ExT) has been shown to reduce sympathetic drive during heart failure (HF). The subfornical organ (SFO) is involved in the neural control of sympathetic drive. We hypothesized that an activated SFO contributes to enhanced sympathetic activity in HF. We also postulated that ExT would reduce the activation of the SFO and its contribution to the sympathetic drive during HF. Sprague-Dawley rats were subjected to coronary artery ligation to induce HF. Rats were assigned to ExT for 3-4 wk. Rats with HF had a 2.5-fold increase in FosB-positive cells in the SFO compared with sham-operated rats, and this was normalized by ExT. Microinjection of ANG II (100 pmol) into the SFO resulted in a greater increase in renal sympathetic nerve activity (RSNA), blood pressure, and heart rate in the HF group than in the sham-operated group. These responses were normalized after ExT (change in RSNA: 23 ± 3% vs. 8 ± 2%). ExT also abolished the decrease in RSNA in HF rats after the microinjection of losartan (200 pmol) into the SFO (-21 ± 4% vs. -2 ± 3%). Finally, there was elevated mRNA (5-fold) and protein expression (43%) of ANG II type 1 receptors in the SFO of rats with HF, which were reversed after ExT. These data suggest that the enhanced activity of the SFO by elevated tonic ANG II contributes to the enhanced sympathoexcitation exhibited in HF. The decrease in ANG II type 1 receptor expression in the SFO by ExT may be responsible for reversing the neuronal activation in the SFO and SFO-mediated sympathoexcitation in rats with HF.

Insulin modulates the electrical activity of subfornical organ neurons

Insulin plays a crucial role in the regulation of energy balance. Within the central nervous system, hypothalamic nuclei such as the arcuate and ventromedial nuclei are targets of insulin; however, insulin may only access these nuclei after transport across the blood-brain barrier. Neurons of the subfornical organ are not protected by the blood-brain barrier and can rapidly detect and respond to circulating hormones such as leptin and ghrelin. Moreover, subfornical organ neurons form synaptic connections with hypothalamic control centers that regulate energy balance, including the arcuate and dorsomedial nuclei. However, it is unknown whether subfornical organ neurons respond to insulin. Using whole-cell current clamp, we examined the electrophysiological effects of insulin on rat subfornical organ neurons. Upon insulin application, 70% of neurons tested were responsive, with 33% of neurons tested (9/27) exhibiting hyperpolarization of membrane potential (-8.7 ± 1.7 mV) and 37% (10/27) exhibiting depolarization (10.5 ± 2.8 mV). Using pharmacological blockade, our data further indicate that the hyperpolarization was mediated by opening of KATP channels, whereas depolarization resulted from opening of Ih channels. These data are the first to show that insulin exerts a direct effect on the electrical activity of subfornical organ neurons and support the notion that the subfornical organ may act to communicate information on circulating satiety signals to homeostatic control centers.

Exercise Training (ExT) Normalizes Subfornical Organ (SFO)‐ Mediated Sympathoexcitation in Chronic Heart Failure (HF)

ExT normalizes sympathoexcitation (SymEx) exhibited in HF, however the underlying mechanisms are not clear. The SFO is involved in dictating SymEx and is influenced by peptides such as angiotensin (Ang) II due to its weak blood‐brain barrier. We hypothesized that neuronal activity in the SFO would be enhanced in HF and contribute to increased Ang II‐mediated SymEx, and that ExT would reverse this process. HF induced by coronary artery ligation in Sprague‐Dawley rats was confirmed by echocardiography 4 weeks after surgery. HF rats displayed increased neuronal activity in the SFO, as assessed by FosB immunohistochemistry (101 ± 9 vs. 29 ± 2 cells). This increase was attenuated after 4 weeks of treadmill ExT (36 ± 7 cells). In anesthetized rats, microinjection of Ang II into the SFO increased renal sympathetic nerve activity (RSNA) to a greater extent in HF (23 ± 3 vs. 9 ± 1%; 100 pmol), and was normalized after ExT (8 ± 2%). ExT also abolished the decrease in RSNA in HF rats after losartan microinjection into the SFO (−21 ± 4 vs. −1 ± 3%; 200 pmol), implying higher endogenous Ang II tone in HF. ExT reversed the elevated mRNA and protein expression of AT1R in the SFO of rats with HF. The enhanced activation of the SFO by elevated tonic Ang II contributes to the enhanced SymEx exhibited in HF. The reduction in AT1 receptors in the SFO by ExT may be responsible for restoring the neuronal activation in the SFO and SFO‐mediated SymEx. Supp. by NIH HL62222.

Dopamine modulates neuronal excitability pre- and post-synaptically in the rat subfornical organ

The aims of this study were to investigate the involvement of dopamine (DA) in drinking behaviour related to body fluid balance. All experiments were performed in rats. Water intake induced by intracerebroventricular injection of angiotensin II (ANGII) was suppressed by co-injection of DA in a dose-dependent manner. RT-PCR revealed the presence of mRNAs for all known DA receptors, D1-D5, in the subfornical organ (SFO), a brain region that plays a key role in regulating drinking behaviour. Extracellular recordings and whole-cell patch-clamp recordings from SFO neurons showed that DA or the D4 selective agonist PD168077 inhibited spontaneous electrical activity. The D4 antagonist L745870 blocked DA-induced inhibition of spontaneous electrical activity in SFO neurons. Under conditions of synaptic blockade, the inhibitory effects of DA and PD168077 still remained, but the D2/D3 agonist quinpirole and the D1/D5 agonist SKF38393 had almost no effect on electrical activity. While DA induced excitation in a small number of neurons, these excitatory responses almost disappeared following synaptic blockade. All neurons with firing rates that were suppressed by DA were excited by ANGII. In voltage clamp mode, we found that DA and quinpirole, but not SKF38393, suppressed GABAergic miniature inhibitory post-synaptic currents. These results suggest that DA inhibits neuronal activity in ANGII-sensitive SFO neurons primarily through the postsynaptic D4 receptor subtype. This may be a cause of the suppression of ANGII-induced water intake by DA. In addition, the inhibitory DA responses in SFO neurons may be modulated by presynaptic suppression of GABAergic inhibitory inputs through D2/D3 receptor subtypes.

Dopamine inhibits neurons in the rat subfornical organ through post-synaptic D4 receptor

Dopamine inhibits neurons in the rat subfornical organ through post-synaptic D4 receptor