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Central infusion of Ang II (angiotensin II) has been associated with increased sympathetic outflow resulting in neurogenic hypertension. In the present study, we appraised whether the chronic increase in central Ang II activates the paraventricular nucleus of the hypothalamus (PVN) resulting in elevated sympathetic tone and altered baro- and chemoreflexes. Further, we evaluated the contribution of HIF-1α (hypoxia-inducible factor-1α), a transcription factor involved in enhancing the expression of N-methyl-D-aspartate receptors and thus glutamatergic-mediated sympathetic tone from the PVN. Ang II infusion (20 ng/minute, intracerebroventricular, 14 days) increased mean arterial pressure (126±9 versus 84±4 mm Hg), cardiac sympathetic tone (96±7 versus 75±6 bpm), and decreased cardiac parasympathetic tone (16±2 versus 36±3 versus bpm) compared with saline-infused controls in conscious rats. The Ang II-infused group also showed an impaired baroreflex control of heart rate (-1.50±0.1 versus -2.50±0.3 bpm/mm Hg), potentiation of the chemoreflex pressor response (53±7 versus 30±7 mm Hg) and increased number of FosB-labeled cells (53±3 versus 19±4) in the PVN. Concomitant with the activation of the PVN, there was an increased expression of HIF-1α and N-Methyl-D-Aspartate-type1 receptors in the PVN. Further, Ang II-infusion showed increased renal sympathetic nerve activity (20.5±2.3% versus 6.4±1.9% of Max) and 3-fold enhanced renal sympathetic nerve activity responses to microinjection of N-methyl-D-aspartate (200 pmol) into the PVN of anesthetized rats. Further, silencing of HIF-1α in NG108 cells abrogated the expression of N-methyl-D-aspartate-N-methyl-D-aspartate-type1 induced by Ang II. Taken together, our studies suggest a novel Ang II-HIF-1α-N-methyl-D-aspartate receptor-mediated activation of preautonomic neurons in the PVN, resulting in increased sympathetic outflow and alterations in baro- and chemoreflexes.

Renal denervation (RDN) has been shown to restore endogenous neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) and reduce sympathetic drive during chronic heart failure (CHF). The purpose of the present study was to assess the contribution of afferent renal nerves to the nNOS-mediated sympathetic outflow within the PVN in rats with CHF. CHF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, selective afferent RDN (A-RDN) was performed by bilateral perivascular application of capsaicin on the renal arteries. Seven days after intervention, nNOS protein expression, nNOS immunostaining signaling, and diaphorase-positive stained cells were significantly decreased in the PVN of CHF rats, changes that were reversed by A-RDN. A-RDN reduced basal lumbar sympathetic nerve activity in rats with CHF (8.5%±0.5% versus 17.0%±1.2% of max). Microinjection of nNOS inhibitor L-NMMA (L-N G -monomethyl arginine citrate) into the PVN produced a blunted increase in lumbar sympathetic nerve activity in rats with CHF. This response was significantly improved after A-RDN (Δ lumbar sympathetic nerve activity: 25.7%±2.4% versus 11.2%±0.9%). Resting afferent renal nerves activity was substantially increased in CHF compared with sham rats (56.3%±2.4% versus 33.0%±4.7%). These results suggest that intact afferent renal nerves contribute to the reduction of nNOS in the PVN. A-RDN restores nNOS and thus attenuates the sympathoexcitation. Also, resting afferent renal nerves activity is elevated in CHF rats, which may highlight a crucial neural mechanism arising from the kidney in the maintenance of enhanced sympathetic drive in CHF.

Accumulated evidence indicates that obesity-induced type 2 diabetes (T2D) is associated with enhanced sympathetic activation. The present study was conducted to investigate the role for leptin-glutamate signaling within the hypothalamus in regulating sympathetic nerve activity. In anesthetized rats, microinjections of leptin (5 ng ~ 100 ng) into the arcuate nucleus (ARCN) and paraventricular nucleus (PVN) induced increases in renal sympathetic nerve activity (RSNA), blood pressure (BP), and heart rate (HR). Prior microinjections of NMDA receptor antagonist AP5 (16 pmol) into the ARCN or PVN reduced leptin-induced increases in RSNA, BP, and HR in both ARCN and PVN. Knockdown of a leptin receptor with siRNA inhibited NMDA-induced increases in RSNA, BP, and HR in the ARCN but not in the PVN. Confocal calcium imaging in the neuronal NG108 and astrocytic C6 cells demonstrated that preincubation with leptin induced an increase in intracellular calcium green fluorescence when the cells were challenged with glutamate. In high-fat diet and low-dose streptozotocin-induced T2D rats, we found that leptin receptor and NMDA NR 1 receptor expressions in the ARCN and PVN were significantly increased. In conclusion, these studies provide evidence that within the hypothalamic nuclei, leptin-glutamate signaling regulates the sympathetic activation. This may contribute to the sympathoexcitation commonly observed in obesity-related T2D.

Background: Increased sympathetic outflow is a major contributor to the progression of chronic heart failure (CHF). Potentiation of glutamatergic tone has been causally related to the sympathoexcitation in CHF. Specifically, an increase in the N-methyl-d-aspartate-type 1 receptor (NMDA-NR 1 ) expression within the paraventricular nucleus (PVN) is critically linked to the increased sympathoexcitation during CHF. However, the molecular mechanism(s) for the upregulation of NMDA-NR 1 remains unexplored. We hypothesized that hypoxia via hypoxia-inducible factor 1α (HIF-1α) might contribute to the augmentation of the NMDA-NR 1 -mediated sympathoexcitatory responses from the PVN in CHF., Methods and Results: Immunohistochemistry staining, mRNA, and protein for hypoxia-inducible factor 1α were upregulated within the PVN of left coronary artery-ligated CHF rats. In neuronal cell line (NG108-15) in vitro, hypoxia caused a significant increase in mRNA and protein for HIF-1α (2-fold) with the concomitant increase in NMDA-NR 1 mRNA, protein levels, and glutamate-induced Ca + influx. Chromatin immunoprecipitation assay identified HIF-1α binding to NMDA-NR 1 promoter during hypoxia. Silencing of HIF-1α in NG108 cells leads to a significant decrease in expression of NMDA-NR 1 , suggesting that expression of HIF-1α is necessary for the upregulation of NMDA-NR 1 . Consistent with these observations, HIF-1α silencing within the PVN abrogated the increased basal sympathetic tone and sympathoexcitatory responses to microinjection of NMDA in the PVN of rats with CHF., Conclusions: These results uncover a critical role for HIF-1 in the upregulation of NMDA-NR 1 to mediate sympathoexcitation in CHF. We conclude that subtle hypoxia within the PVN may act as a metabolic cue to modulate sympathoexcitation during CHF., (© 2016 American Heart Association, Inc.)

Renal denervation (RDN) has been postulated to reduce sympathetic drive during heart failure (HF), but the central mechanisms are not completely understood. The purpose of the present study was to assess the contribution of neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN) in modulating sympathetic outflow in rats with HF that underwent RDN. HF was induced in rats by ligation of the left coronary artery. Four weeks after surgery, bilateral RDN was performed. Rats with HF had an increase in FosB-positive cells in the PVN with a concomitant increase in urinary excretion of norepinephrine, and both of these parameters were ameliorated after RDN. nNOS-positive cells immunostaining, diaphorase staining, and nNOS protein expression were significantly decreased in the PVN of HF rats, findings that were ameliorated by RDN. Microinjection of nNOS inhibitor N(G)-monomethyl l-arginine into the PVN resulted in a blunted increase in lumbar sympathetic nerve activity (11±2% versus 24±2%) in HF than in sham group. This response was normalized after RDN. Stimulation of afferent renal nerves produced a greater activation of PVN neurons in rats with HF. Afferent renal nerve stimulation elicited a greater increase in lumbar sympathetic nerve activity in rats with HF than in sham rats (45±5% versus 22±2%). These results suggest that intact renal nerves contribute to the reduction of nNOS in the PVN, resulting in the activation of the neurons in the PVN of rats with HF. RDN restores nNOS and thus attenuates the sympathoexcitation commonly observed in HF., (© 2016 American Heart Association, Inc.)

Renal denervation for the treatment of hypertension has proven to be successful; however, the underlying mechanism/s are not entirely clear. To determine if preautonomic neurons in the paraventricular nucleus (PVN) respond to afferent renal nerve (ARN) stimulation, extracellular single-unit recording was used to investigate the contribution of the rostral ventrolateral medulla (RVLM)-projecting PVN (PVN-RVLM) neurons to the response elicited during stimulation of ARN. In 109 spontaneously active neurons recorded in the PVN of anesthetized rats, 25 units were antidromically activated from the RVLM. Among these PVN-RVLM neurons, 84% (21/25) were activated by ARN stimulation. The baseline discharge rate was significantly higher in these neurons than those PVN-RVLM neurons not activated by ARN stimulation (16%, 4/25). The responsiveness of these neurons to baroreflex activation induced by phenylephrine and activation of cardiac sympathetic afferent reflex (CSAR) was also examined. Almost all of the PVN neurons that responded to ARN stimulation were sensitive to baroreflex (95%) and CSAR (100%). The discharge characteristics for nonevoked neurons (not activated by RVLM antidromic stimulation) showed that 23% of these PVN neurons responded to ARN stimulation. All the PVN neurons that responded to ARN stimulation were activated by N-methyl-D-aspartate, and these responses were attenuated by the glutamate receptor blocker AP5. These experiments demonstrated that sensory information originating in the kidney is integrated at the level of preautonomic neurons within the PVN, providing a novel mechanistic insight for use of renal denervation in the modulation of sympathetic outflow in disease states such as hypertension and heart failure., (Copyright © 2015 the American Physiological Society.)

The study was conducted to investigate the role for dopamine in the centrally mediated sympathoexcitatory response in rats with Type 2 diabetes (T2D). T2D was induced by a combination of high-fat diet (HFD) and low-dose streptozotocin (STZ). HFD/STZ treatment for 12-14 wk resulted in significant increase in the number of FosB-positive cells in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM). In anesthetized rats, administration of exogenous dopamine (dopamine hydrochloride, 20 mM) in the PVN, but not in the RVLM, elicited decreases in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in control rats and but not in the T2D rats. Blocking the endogenous dopamine with dopamine D1/D5 receptor antagonist SCH39166 (2 mM) in the PVN and RVLM, resulted in increases in RSNA, MAP, and heart rate (HR) in both control and T2D rats. These responses were significantly attenuated in T2D rats compared with control rats (PVN - ΔRSNA: 21 ± 10 vs. 44 ± 2%; ΔMAP: 7 ± 3 vs. 19 ± 6 mmHg, ΔHR: 17 ± 5 vs. 32 ± 4 bpm, P < 0.05). There were no significant increases in response to dopamine D2/D3 receptor antagonist raclopride application in the PVN and RVLM of both control and T2D rats. Furthermore, there were decreased dopamine D1 receptor and D2 receptor expressions in the PVN of T2D rats. Taken together, these data suggest that reduced endogenous dopaminergic tone within the PVN may contribute to the sympathoexcitation in T2D.

Exercise training (ExT) normalizes the increased sympathetic outflow in chronic heart failure (HF). The underlying mechanisms are not clearly understood. We hypothesized that ExT normalized the blunted central component of the baroreflex control of renal sympathetic nerve activity (RSNA) in HF. Four groups of rats [sham operated (sham)-sedentary (Sed), sham-ExT, HF-Sed, and HF-ExT] were used. HF was induced by left coronary artery ligation, and ExT consisted of 3 wk of treadmill running. In anesthetized rats, the decrease in RSNA in response to aortic depressor nerve stimulation (5-40 Hz) in the HF-Sed group was significantly lower than that in the sham-Sed group (-37 ± 7% vs. -63 ± 8% at 40 Hz, P < 0.05). In the HF-ExT group, responses in RSNA, mean arterial pressure (MAP), and heart rate (HR) were not significantly different from those in the sham-Sed or sham-ExT groups. ExT normalized blunted RSNA, MAP, and HR responses to bicuculline microinjections into the paraventricular nucleus (PVN) in rats with HF. Activation of the PVN by blockade of GABA receptors with bicuculline in normal control rats blunted the centrally component of the baroreflex arc. GABAA-α1 and -β1 receptor protein expression were significantly lower (by 48% and 30%) in the HF-Sed group, but ExT normalized this difference between the HF and sham groups. These data suggest that one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of central integrative mechanisms, perhaps via improving the inhibitory GABAergic mechanism within the PVN, on the baroreflex arc.

Exercise training (ExT) normalizes the increased sympathetic outflow in heart failure (HF), but the underlying mechanisms are not known. We hypothesized ExT would normalize the augmented activation of the paraventricular nucleus (PVN) via an angiotensinergic mechanism during HF. Four groups of rats used were the following: 1) sham-sedentary (Sed); 2) sham-ExT; 3) HF-Sed, and 4) HF-ExT. HF was induced by left coronary artery ligation. Four weeks after surgery, 3 wk of treadmill running was performed in ExT groups. The number of FosB-positive cells in the PVN was significantly increased in HF-Sed group compared with the sham-Sed group. ExT normalized (negated) this increase in the rats with HF. In anesthetized condition, the increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in response to microinjection of angiotensin (ANG) II (50∼200 pmol) in the PVN of HF-Sed group were significantly greater than of the sham-Sed group. In the HF-ExT group the responses to microinjection of ANG II were not different from sham-Sed or sham-ExT groups. Blockade of ANG II type 1 (AT(1)) receptors with losartan in the PVN produced a significantly greater decrease in RSNA, MAP, and HR in HF-Sed group compared with sham-Sed group. ExT prevented the difference between HF and sham groups. AT(1) receptor protein expression was increased 50% in HF-Sed group compared with sham-Sed group. In the HF-ExT group, AT(1) receptor protein expression was not significantly different from sham-Sed or sham-ExT groups. In conclusion, one mechanism by which ExT alleviates elevated sympathetic outflow in HF may be through normalization of angiotensinergic mechanisms within the PVN.

The paraventricular nucleus (PVN) of the hypothalamus is involved in the neural control of sympathetic drive, but the precise mechanism(s) that influences the PVN is not known. The activation of the PVN may be influenced by input from higher forebrain areas, such as the median preoptic nucleus (MnPO) and the subfornical organ (SFO). We hypothesized that activation of the MnPO or SFO would drive the PVN through a glutamatergic pathway. Neuroanatomical connections were confirmed by the recovery of a retrograde tracer in the MnPO and SFO that was injected bilaterally into the PVN in rats. Microinjection of 200 pmol of N-methyl-d-aspartate (NMDA) or bicuculline-induced activation of the MnPO and increased renal sympathetic activity (RSNA), mean arterial pressure, and heart rate in anesthetized rats. These responses were attenuated by prior microinjection of a glutamate receptor blocker AP5 (4 nmol) into the PVN (NMDA - ΔRSNA 72 ± 8% vs. 5 ± 1%; P < 0.05). Using single-unit extracellular recording, we examined the effect of NMDA microinjection (200 pmol) into the MnPO on the firing activity of PVN neurons. Of the 11 active neurons in the PVN, 6 neurons were excited by 95 ± 17% (P < 0.05), 1 was inhibited by 57%, and 4 did not respond. The increased RSNA after activation of the SFO by ANG II (1 nmol) or bicuculline (200 pmol) was also reduced by AP5 in the PVN (for ANG II - ΔRSNA 46 ± 7% vs. 17 ± 4%; P < 0.05). Prior microinjection of ANG II type 1 receptor blocker losartan (4 nmol) into the PVN did not change the response to ANG II or bicuculline microinjection into the SFO. The results from this study demonstrate that the sympathoexcitation mediated by a glutamatergic mechanism in the PVN is partially driven by the activation of the MnPO or SFO.

Angiotensin (ANG)-converting enzyme (ACE)2 in brain regions such as the paraventricular nucleus (PVN) controlling cardiovascular function may be involved in the regulation of sympathetic outflow in chronic heart failure (CHF). The purpose of this study was to determine if ACE2 plays a role in the central regulation of sympathetic outflow by regulating neuronal nitric oxide (NO) synthase (nNOS) in the PVN. We investigated ACE2 and nNOS expression within the PVN of rats with CHF. We then determined the effects of ACE2 gene transfer in the PVN on the contribution of NO-mediated sympathoinhibition in rats with CHF. The results showed that there were decreased expressions for ACE2, the ANG-(1-7) receptor, and nNOS within the PVN of rats with CHF. After the application of adenovirus vectors encoding ACE2 (AdACE2) into the PVN, the increased expression of ACE2 in the PVN was confirmed by Western blot analysis. AdACE2 transfection significantly increased nNOS protein levels (change of 50 ± 5%) in the PVN of CHF rats. In anesthetized rats, AdACE2 treatment attenuated the responses of renal sympathetic nerve activity (RSNA), mean arterial pressure, and heart rate to the NOS inhibitor N-monomethyl-L-arginine in rats with CHF (RSNA: 28 ± 3% vs. 16 ± 3%, P < 0.05) compared with CHF + AdEGFP group. Furthermore, neuronal NG-108 cells incubated with increasing doses of AdACE2 showed a dose-dependent increase in nNOS protein expression (60% at the highest dose). Taken together, our data highlight the importance of increased expression and subsequent interaction of ACE2 and nNOS within the PVN, leading to a reduction in sympathetic outflow in the CHF condition.