Increase In Lumbar Sympathetic Nerve Activity Research Articles

Orexin receptors in paraventricular nucleus influence sexual behavior via regulating the sympathetic outflow in males.

Orexins are hypothalamic neuropeptides associated with various neurophysiological activities such as sleep, arousal, and reward. However, there are few studies investigating the relationships between orexin receptors in the paraventricular nucleus and sexual behaviors. To explore the roles of orexin receptors in the paraventricular nucleus on sexual behaviors and uncover its potential mechanisms in males. Orexin A, orexin 1 receptor antagonist SB334867, and orexin 2 receptor antagonist TCS-OX2-29 were microinjected into the paraventricular nucleus to investigate the effects of orexin receptors on copulatory behavior testing of C57BL/6 mice. To explore if ejaculation could activate orexin 1 receptor-expressing neurons in the paraventricular nucleus, fluorescence immunohistochemical double staining was utilized. The levels of serum norepinephrine were measured and the lumbar sympathetic nerve activity was recorded to reflect the sympathetic nervous system activity. Moreover, the bulbospongiosus muscle-electromyogram was recorded and analyzed. To test whether perifornical/lateral hypothalamic area orexinergic neurons directly projected to the paraventricular nucleus, virus retrograde tracing technology was utilized. Orexin A significantly enhanced sexual performance by shortening the intromission and ejaculation latencies, and increasing the mount and intromission frequencies, while the opposite outcomes appeared with SB334867. However, TCS-OX2-29 had no significant effects on sexual behaviors. Moreover, orexin A increased lumbar sympathetic nerve activity and the levels of serum norepinephrine, while SB334867 decreased lumbar sympathetic nerve activity and norepinephrine, which caused a significant decrease in sympathetic nervous system outflow. Meanwhile, a robust increase in the bulbospongiosus muscle-electromyogram activity was identified after microinjecting orexin A. Furthermore, cFos immunopositive cells were increased and double stained with orexin 1 receptor-expressing neurons in the mating group. Additionally, the retrograde tracing results demonstrated that orexinergic neurons in the perifornical/lateral hypothalamic area directly projected to the paraventricular nucleus. Orexin 1 receptor in the paraventricular nucleus could influence the ejaculatory reflex via mediating the sympathetic nervous system activity, which might be of great importance in the treatment of premature ejaculation in the future.

Effects of Psidium guajava L. leaves extract on blood pressure control and IL-10 production in salt-dependent hypertensive rats

Psidium guajava (guava) leaves extract displays anti-hypertensive properties by mechanisms not yet fully understood. Here, we investigated whether sympathetic drive and immune signaling mechanisms are involved with the antihypertensive effect of the guava extract in a model of salt-dependent hypertension. Raw guava extract (rPsE) was characterized by colorimetric and UPLC-MS techniques. Two doses of rPsE (100 and 200 mg/kg) were evaluated for anti-hypertensive effect using a suspension system (PsE). Weaned male Wistar rats were put on a high-salt diet (HSD, 0.90 % Na+) for 16 weeks and received gavages of PsE for the last 4 weeks. Blood pressure (BP) was measured at the end of treatment in conscious rats. The neurogenic pressor effect was assessed by ganglionic blockade with hexamethonium. Autonomic modulation of heart rate was evaluated by spectral analysis. The effects of orally administered PsE on lumbar sympathetic nerve activity (LSNA) were assessed in anesthetized rats. Blood IL-10, IL-17A, and TNF were measured. The increased neurogenic pressor effect of HSD rats was reduced by PsE 100 mg/kg, but not by 200 mg/kg. PsE (200 mg/kg) administration in anesthetized rats produced a greater fall in BP of HSD rats compared to standard salt diet (SSD) rats. PsE hypotensive response elicited an unproportionable increase in LSNA of HSD rats compared to SSD rats. PsE (200 mg/kg) increased plasma concentrations of IL-10 but had no effect on TNF or IL-17A. Our data indicate that the antihypertensive effects of PsE may involve autonomic mechanisms and immunomodulation by overexpression of IL-10 in salt-dependent hypertensive rats.

The Effects of Pregnancy on the Sympathoexcitatory and Pressor Responses to Nanoinjection of Angiotensin II into the Arcuate Nucleus

Pregnancy induces profound increases in sympathetic nerve activity (SNA). Angiotensin II (AngII) may contribute, since systemic or central blockade of AngII production or receptors (AT1aR) decreases SNA in pregnant animals. However, the brain sites involved are unknown. One possibility is the arcuate nucleus (ArcN), since AngII binds to ArcN AT1aR to increase SNA, and the ArcN is a well‐established reproductive integrating center. To begin to test this hypothesis, we measured the changes in SNA and mean arterial pressure (MAP) in response to bilateral nanoinjections of AngII (30 pmol in 30 nL) into the ArcN of α‐chloralose anesthetized pregnant rats and virgin rats throughout the estrus cycle. ArcN AngII slowly increased (P<0.05) lumbar SNA (LSNA) in pregnant rats (by 50±10 % after 90 min; n=6), and the rise was larger than in proestrus rats (‐1±8 %; n=5; P<0.05), similar to diestrus rats (40±9 %; n=4), but less than estrus rats (72±12 %; n=6; P<0.05). ArcN AngII rapidly increased (P<0.05) MAP by ~10 mmHg (after 10 min) in all groups, but after 90 min the increase was sustained (P<0.05) only in pregnant (13±2 mmHg) and estrus (11±4 mmHg) rats. A cohort of ArcN neurons projects to and stimulates vasopressinergic neurons in the hypothalamic paraventricular and supraoptic nuclei. Therefore, to test if the initial or sustained increases in MAP were mediated by vasopressin, another set of estrus and pregnant rats were pretreated with the Manning Compound (V1x; 5 μg iv) 15 min before ArcN AngII. In estrus rats (n=4), V1x had no effects on baseline LSNA or MAP, but completely blocked the initial rise in MAP induced by ArcN AngII (2±3 mmHg). Nevertheless, the increase in MAP at 90 min was unchanged (13±3 mmHg; P<0.05). The LSNA response to ArcN AngII (83±9 %) was also not altered. In pregnant rats pretreated with V1x (n=4), baseline MAP decreased (P<0.05) by 10±1 mmHg, and again ArcN AngII failed to increase MAP initially (2±1 mmHg). However, the increase in MAP 90 min after ArcN AngII (7±2 mmHg) was the same as the increase 90 min after injection of artificial cerebrospinal fluid into the ArcN of V1x‐treated pregnant rats (5±2 mmHg). Again, the increase in LSNA to ArcN AngII was unchanged (30±6 %). Finally, RNAScope was used to assay AT1aR mRNA expression in the ArcN in diestrus, proestrus, and pregnant rats. ArcN AT1aR were undetectable in proestrus rats and were expressed at low but detectable levels in diestrus rats. In contrast, pregnancy markedly increased the expression of AT1aR in the ArcN. In summary, during pregnancy, ArcN AngII produces substantial increases in LSNA in association with elevated AT1aR expression. Moreover, the increase in MAP in response to ArcN AngII was reduced or eliminated in estrus and pregnant rats pretreated with a vasopressin V1 receptor blocker. In conclusion, these data highlight the ArcN as a potential site at which AngII increases SNA during normal pregnancy. The data also suggest that AngII can elicit an increase in vasopressin secretion via an action in the ArcN and that vasopressin contributes to the pressor responses induced by ArcN AngII in both estrus and pregnant rats.Support or Funding InformationHL128181

Sites and sources of sympathoexcitation in obese male rats: role of brain insulin.

In males, obesity increases sympathetic nerve activity (SNA), but the mechanisms are unclear. Here, we investigate insulin, via an action in the arcuate nucleus (ArcN), and downstream neuropathways, including melanocortin receptor 3/4 (MC3/4R) in the hypothalamic paraventricular nucleus (PVN) and dorsal medial hypothalamus (DMH). We studied conscious and α-chloralose-anesthetized Sprague-Dawley rats fed a high-fat diet, which causes obesity prone (OP) rats to accrue excess fat and obesity-resistant (OR) rats to maintain fat content, similar to rats fed a standard control (CON) diet. Nonspecific blockade of the ArcN with muscimol and specific blockade of ArcN insulin receptors (InsR) decreased lumbar SNA (LSNA), heart rate (HR), and mean arterial pressure (MAP) in OP, but not OR or CON, rats, indicating that insulin supports LSNA in obese males. In conscious rats, intracerebroventricular infusion of insulin increased MAP only in OP rats and also improved HR baroreflex function from subnormal to supranormal. The brain sensitization to insulin may elucidate how insulin can drive central SNA pathways when transport of insulin across the blood-brain barrier may be impaired. Blockade of PVN, but not DMH, MC3/4R with SHU9119 decreased LSNA, HR, and, MAP in OP, but not OR or CON, rats. Interestingly, nanoinjection of the MC3/4R agonist melanotan II (MTII) into the PVN increased LSNA only in OP rats, similar to PVN MTII-induced increases in LSNA in CON rats after blockade of sympathoinhibitory neuropeptide Y Y1 receptors. ArcN InsR expression was not increased in OP rats. Collectively, these data indicate that obesity increases SNA, in part via increased InsR signaling and downstream PVN MC3/4R.

Resistance to the sympathoexcitatory effects of insulin and leptin in late pregnant rats.

Pregnancy increases sympathetic nerve activity (SNA), although the mechanisms responsible for this remain unknown. We tested whether insulin or leptin, two sympathoexcitatory hormones increased during pregnancy, contribute to this. Transport of insulin across the blood-brain barrier in some brain regions, and into the cerebrospinal fluid (CSF), was increased, although brain insulin degradation was also increased. As a result, brain and CSF insulin levels were not different between pregnant and non-pregnant rats. The sympathoexcitatory responses to insulin and leptin were abolished in pregnant rats. Blockade of arcuate nucleus insulin receptors did not lower SNA in pregnant or non-pregnant rats. Collectively, these data suggest that pregnancy renders the brain resistant to the sympathoexcitatory effects of insulin and leptin, and that these hormones do not mediate pregnancy-induced sympathoexcitation. Increased muscle SNA stimulates glucose uptake. Therefore, during pregnancy, peripheral insulin resistance coupled with blunted insulin- and leptin-induced sympathoexcitation ensures adequate delivery of glucose to the fetus. Pregnancy increases basal sympathetic nerve activity (SNA), although the mechanism responsible for this remains unknown. Insulin and leptin are two sympathoexcitatory hormones that increase during pregnancy, yet, pregnancy impairs central insulin- and leptin-induced signalling. Therefore, to test whether insulin or leptin contribute to basal sympathoexcitation or, instead, whether pregnancy induces resistance to the sympathoexcitatory effects of insulin and leptin, we investigated α-chloralose anaesthetized late pregnant rats, which exhibited increases in lumbar SNA (LSNA), splanchnic SNA and heart rate (HR) compared to non-pregnant animals. In pregnant rats, transport of insulin into cerebrospinal fluid and across the blood-brain barrier in some brain regions increased, although brain insulin degradation was also increased; brain and cerebrospinal fluid insulin levels were not different between pregnant and non-pregnant rats. Although i.c.v. insulin increased LSNA and HR and baroreflex control of LSNA and HR in non-pregnant rats, these effects were abolished in pregnant rats. In parallel, pregnancy completely prevented the actions of leptin with respect to increasing lumbar, splanchnic and renal SNA, as well as baroreflex control of SNA. Blockade of insulin receptors (with S961) in the arcuate nucleus, the site of action of insulin, did not decrease LSNA in pregnant rats, despite blocking the effects of exogenous insulin. Thus, pregnancy is associated with central resistance to insulin and leptin, and these hormones are not responsible for the increased basal SNA of pregnancy. Because increases in LSNA to skeletal muscle stimulates glucose uptake, blunted insulin- and leptin-induced sympathoexcitation reinforces systemic insulin resistance, thereby increasing the delivery of glucose to the fetus.

Sex differences in the sympathoexcitatory response to insulin in obese rats: role of neuropeptide Y.

Intracerebroventricular insulin increased sympathetic nerve activity (SNA) and baroreflex control of SNA and heart rate more dramatically in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced lumbar SNA (LSNA) responses were associated with reduced tonic inhibition of LSNA by neuropeptide Y (NPY) in the PVN. However, PVN NPY injection decreased LSNA similarly in obesity prone/obesity resistant/control rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the arcuate nucleus became resistant to the inhibitory effects of insulin. A high-fat diet did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced 'selective sensitization' of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. Obesity increases sympathetic nerve activity (SNA) in men but not women; however, the mechanisms are unknown. We investigated whether intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high-fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane anaesthesia. The LSNA response was magnified in OP males but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males as a result of a decrease in NPY inputs. By contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on the sympathoexcitatory actions of insulin and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.

Airway obstruction produces widespread sympathoexcitation: role of hypoxia, carotid chemoreceptors, and NTS neurotransmission.

Obstructive sleep apnea (OSA) is the most common respiratory disturbance of sleep and is closely associated to cardiovascular diseases. In humans, apnea increases respiratory effort and elevates muscle sympathetic nerve activity (SNA), but the primary stimulus for the SNA activation has not been identified. We recently developed a model of apnea in rodents using acute airway obstruction. In this study, we employed this model to test whether the elevation in SNA was mediated by hypoxia, carotid chemoreceptors, or neurotransmission in the nucleus tractus solitarius (NTS). In anesthetized, male Sprague–Dawley rats, airway obstruction (20s) increased phrenic nerve activity (PNA), arterial blood pressure (ABP), and lumbar, renal, and splanchnic SNA. The changes in SNA were similar across all three sympathetic nerves. Inactivation of chemoreceptors by hyperoxia (100% O2) or surgical denervation of carotid chemoreceptors attenuated, but did not eliminate, the changes in SNA and ABP produced by airway obstruction. To interrupt afferent information from carotid chemoreceptor and extracarotid afferents to the hindbrain, airway obstruction was performed before and after NTS microinjection of the GABAA agonist muscimol or a cocktail of NMDA and non‐NMDA antagonists. Inhibition of NTS neurons or blockade of glutamatergic receptors attenuated the increase in lumbar SNA, splanchnic SNA, renal SNA, and PNA. Collectively, these findings suggest that PNA and SNA responses induced by airway obstruction depend, in part, on chemoreceptors afferents and glutamatergic neurotransmission in the NTS.

Abstract P391: Local Ionotropic Glutamate Receptors Contribute to the Sympathoexcitatory Effects of Leptin in the Paraventricular Nucleus

In male rats, intracerebroventricular infusion of leptin increases sympathetic nerve activity (SNA), and this response can be partially reversed by nanoinjections of the ionotropic glutamate receptor (iGluR) blocker, kynurenate (KYN), and also the melanocortin type 3/4 receptor (MC3/4R) antagonist, SHU9119, into the hypothalamic paraventricular nucleus (PVN). These results suggest that leptin increases SNA in part via activation of PVN iGluR and MC3/4R. More recently, we found that injection of leptin directly into the PVN also dose-dependently increases SNA. However, the role of PVN iGluR or MC3/4R in this response is unknown. Therefore, we determined if PVN KYN (2.7 nmol in 60 nL) or SHU9119 (30 pmol) reverses the increases in lumbar SNA (LSNA), mean arterial pressure (MAP), and heart rate (HR) evoked by bilateral PVN nanoinjections of leptin (30 ng in 60 nL; n=6) or, as a control, of artificial cerebrospinal fluid (aCSF; n=4). After ninety min, PVN leptin increased (all P<0.05) LSNA to 164±10 % of control, MAP from 106±5 to 118±5 mmHg, and HR from 360±12 to 399±16 bpm. Then, PVN injections of KYN decreased (all P<0.05) LSNA to 133±8 % control, MAP to 108±4 mmHg, and HR to 379±16 bpm. On the other hand, neither PVN aCSF injections (LSNA, to 105±3 % control; MAP, from 107±8 to 109±7 mmHg; and HR, from 363±31 to 367±20 bpm) nor subsequent PVN KYN injections (LSNA, to 102±5 % control; MAP, to 108±6 mmHg; and HR, to 373±17 bpm) significantly altered these variables. In a second set of animals, while PVN leptin (n=4) elicited similar increases (all P<0.05) in LSNA (to 158±8 % control), MAP (from 101±6 to 113±4 mmHg), and HR (from 367±19 to 405±11 bpm), subsequent SHU9119 had no effects (LSNA, to 151±7 % control; MAP, to 108±5 mmHg; and HR, to 400±14 bpm). Again, no changes in these variables were observed in response to PVN injections of aCSF (n=4), even when followed by SHU9119 (LSNA, to 114±11 and then 110±13 % control; MAP, from 107±7 to 109±8 and then 106±9 mmHg; and HR, 357±31 to 355±24 and then 354± 25 bpm). In conclusion, activation of local iGluR, but not MC3/4R, contribute to the increases in LSNA, MAP, and HR in response to PVN leptin.

Abstract 083: Neurons in the Organum Vasculosum of the Lamina Terminalis Contribute to Salt-sensitive Hypertension

Excess dietary salt intake raises plasma and cerebrospinal fluid NaCl concentrations to elevate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Changes in extracellular NaCl concentrations are sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT) - a circumventricular organ that lacks a complete blood-brain barrier. The purpose of the present study was to investigate the hypothesis that salt-sensitive hypertension was mediated, in part, by an elevated activity of OVLT neurons. Dahl-Salt-Sensitive or Sprague-Dawley rats (8-10 weeks) were fed 0.5% or 4.0% NaCl diets for 3-4 weeks. First, in vivo single-unit recordings demonstrate the discharge of OVLT neurons in Dahl-Salt-Sensitive rats was higher after a 4.0% versus 0.5% NaCl diet (4.1±0.4 Hz vs 1.9±0.3 Hz, n=6 per group, P<0.05). OVLT neuronal discharge of Sprague-Dawley rats was not different after a 4.0% or 0.5% NaCl diet (2.1±0.4 Hz vs 1.7±0.3 Hz, n=6-9 per group, P>0.5). In a second set of experiments, injection of hypertonic NaCl (1.0M NaCl, 20nL) into the OVLT produced significantly greater increases in lumbar SNA (131±6% vs 116±3%, n=4 per group, P<0.05) and mean ABP (14±2 vs 8±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl respectively. Sprague-Dawley rats fed 4.0% versus 0.5% NaCl exhibited responses of smaller magnitude for both lumbar SNA (115±4 vs 108±3%, n=4 per group, P<0.05) and mean ABP (9±2 vs 6±2 mmHg, n=4 per group, P<0.05). Interestingly, the duration of the response was much longer in Dahl-Salt-Sensitive versus Sprague-Dawley rats (data not shown). Finally, inhibition of neuronal activity by injection of the GABA agonist muscimol (5mM, 20nL) into the OVLT produced a significantly greater fall in lumbar SNA (-25±4% vs -11±3%, n=4 per group, P<0.05) and mean ABP (-19±4 vs -6±2 mmHg, n=4 per group, P<0.05) of Dahl-Salt-Sensitive rats fed 4.0% versus 0.5% NaCl, respectively. Injection of muscimol into the OVLT of Sprague-Dawley rats did not significantly affect SNA or mean ABP. Collectively, these findings suggest a high salt diet increases the activity of OVLT neurons to elevate SNA and ABP in salt-sensitive hypertension.

NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.

Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi-osmotic mannitol/sorbitol did not alter any variable. In vitro whole-cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi-osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi-osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl- versus osmo-sensing. Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi-osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague-Dawley rats. First, intracerebroventricular (icv) infusion (5μl/10min) of 1.0m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0osmoll-1 ). Second, OVLT microinjection (20nl) of 1.0m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi-osmotic sorbitol did not alter any variable. Third, in vitro whole-cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5mm) and mannitol (+15mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single-unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration-dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi-osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi-osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.

Specific Afferent Renal Denervation Improves Reduced nNOS in the Paraventricular Nucleus in Rats with Heart Failure

Renal denervation (RDN) has been shown to restore endogenous neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN), and at the same time reduce sympathetic drive during heart failure (HF), however the role of afferent versus efferent renal nerves in this response remains to be determined. The purpose of the present study was to assess the contribution of afferent renal nerves (ARN) in the nNOS within the PVN mediated sympathetic outflow in rats with HF. HF 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, A‐RDN was verified by lack of calcitonin gene‐related peptide in renal pelvic wall. A‐RDN reduced basal lumbar sympathetic nerve activity (LSNA) in rats with HF (8.5 ± 0.5 vs 17.0 ± 1.2 % of Max). nNOS‐positive cells immunostaining and ‐diaphorase staining were significantly decreased in the PVN of HF rats, findings that were ameliorated by A‐RDN. Microinjection of nNOS inhibitor L‐NMMA into the PVN resulted in a blunted increase in LSNA in HF, this response was improved after A‐RDN (ΔLSNA: 25.7 ± 2.4 vs 11.2 ± 0.9%). Resting ARN activity, expressed as a percent of peak ARN activity, was substantially increased in HF compared to sham rats (60.9 ± 2.2 vs 27.7 ± 4.4 %Amax). These results suggest that intact ARN contribute to the reduction of nNOS in the PVN, resulting in activation of the neurons in the PVN of rats with HF. Specific A‐RDN restores nNOS and thus attenuates the sympathoexcitation. Also, resting ARN activity is elevated in HF rats, which may highlight a crucial neural mechanism initiated from the kidney in the maintenance of enhanced sympathetic drive in HF.Support or Funding InformationThis work was supported by NIH grant P01 HL62222.

Organum Vasculosum of the Lamina Terminalis Detects NaCl to Elevate Sympathetic Nerve Activity and Blood Pressure.

High-salt diet elevates NaCl concentrations in the cerebrospinal fluid to increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. The organum vasculosum of the lamina terminalis (OVLT) resides along the rostral wall of the third ventricle, lacks a complete blood-brain barrier, and plays a pivotal role in body fluid homeostasis. Therefore, the present study used a multifaceted approach to examine whether OVLT neurons of Sprague-Dawley rats are intrinsically sensitive to changes in extracellular NaCl concentrations and mediate the sympathoexcitatory responses to central NaCl loading. Using in vitro whole-cell recordings, step-wise increases in extracellular NaCl concentrations (2.5-10 mmol/L) produced concentration-dependent excitation of OVLT neurons. Additionally, these excitatory responses were intrinsic to OVLT neurons because hypertonic NaCl evoked inward currents, despite pharmacological synaptic blockade. In vivo single-unit recordings demonstrate that the majority of OVLT neurons (72%, 13/19) display concentration-dependent increases in neuronal discharge to intracarotid (50 μL/15 s) or intracerebroventricular infusion (5 μL/10 minutes) of hypertonic NaCl. Microinjection of hypertonic NaCl (30 nL/60 s) into the OVLT, but not adjacent areas, increased lumbar SNA, adrenal SNA, and arterial blood pressure in a concentration-dependent manner. Renal SNA decreased and splanchnic SNA remained unaffected. Finally, local inhibition of OVLT neurons with the GABAA receptor agonist muscimol (24 nL/10 s) significantly attenuated the sympathoexcitatory and pressor responses to intracerebroventricular infusion of 0.5 mol/L or 1.0 mol/L NaCl. Collectively, these findings indicate that OVLT neurons detect changes in extracellular NaCl concentrations to selectively alter SNA and raise arterial blood pressure.

Abstract 072: Brain Epithelial Sodium Channels on Organum Vasculosum of the Lamina Terminalis Neurons Mediate Sympathoexcitatory Pressor Responses and Neuronal Excitation by Hypertonic NaCl

High dietary salt intake raises cerebrospinal fluid (CSF) [Na + ] in salt sensitive subjects to elevate sympathetic nerve activity (SNA), and arterial blood pressure (ABP). This occurs through excitation of NaCl-sensitive sites in the brain, including the organum vasculosum of the lamina terminalis (OVLT). Intriguingly, intracerebroventricular (ICV) pretreatment with benzamil (a non-voltage gated Na + channel blocker) also attenuates those systemic responses to central NaCl. Thus, I hypothesized that benzamil acts on NaCl-sensitive OVLT neurons to attenuate neuronal excitation and elevated SNA and ABP in response to hypertonic NaCl. To evaluate this hypothesis, lumbar SNA and ABP were measured in anesthetized adult rats in response to ICV infusion of 0.15, 0.5, and 1.0 M NaCl with and without prior OVLT microinjection of benzamil (5nmol per 20nL). ICV infusion of NaCl produced concentration-dependent increases in lumbar SNA (0.15M: 101±3%, 0.5M: 118±3%, 1.0M: 130±9% n=4 per group, P<0.05) and mean ABP (0.15M: 1±1mmHg; 0.5M: 5±1mmHg, 1.0M: 12±2mmHg). OVLT microinjection of benzamil significantly attenuated the increase in lumbar SNA (0.15M: 100±2%, 0.5M: 108±2%, 1.0M: 115±3% n=4 per group, P<0.05) and mean ABP (0.15M: 1±0mmHg; 0.5M: 2±1mmHg, 1.0M: 6±2mmHg). In a parallel set of experiments, in vitro whole-cell recordings of OVLT neurons in slices were performed to assess whether blockade of epithelial sodium channels (ENaC) attenuated NaCl-induced excitation. Bath application of +7.5mM NaCl increase action potential (AP) discharge of OVLT neurons (n=11) from 0.46±0.16 Hz to 1.09±0.26 Hz (P<0.05). Subsequent addition of an ENaC selective concentration of benzamil (0.5μM) reversed AP discharge (0.75±0.24 Hz, P<0.05). Isotonic 0.5μM benzamil did not significantly change NaCl-sensitive OVLT neuron (n=13) AP discharge from baseline (0.54±0.14 Hz to 0.60±0.16 Hz, P>0.05). Collectively, this data indicates that elevations in CSF NaCl concentrations excite OVLT neurons via ENaC to elevate SNA and ABP.

Insulin increases sympathetic nerve activity in part by suppression of tonic inhibitory neuropeptide Y inputs into the paraventricular nucleus in female rats.

Following binding to receptors in the arcuate nucleus (ArcN), insulin increases sympathetic nerve activity (SNA) and baroreflex control of SNA via a pathway that includes the paraventricular nucleus of the hypothalamus (PVN). Previous studies in males indicate that the sympathoexcitatory response is mediated by α-melanocyte stimulating hormone (α-MSH), which binds to PVN melanocortin type 3/4 receptors (MC3/4R). The present study was conducted in α-chloralose-anesthetized female rats to test the hypothesis that suppression of inhibitory neuropeptide Y (NPY) inputs to the PVN is also involved. In support of this, blockade of PVN NPY Y1 receptors with BIBO 3304 (NPY1x), ArcN insulin nanoinjections, and PVN NPY1x followed by ArcN insulin each increased lumbar SNA (LSNA) and its baroreflex regulation similarly. Moreover, prior PVN injections of NPY blocked the sympathoexcitatory effects of ArcN insulin. Finally, PVN nanoinjections of the MC3/4R inhibitor SHU9119 prevented both the acute (15 min) and longer, more slowly developing (60 min), increases in LSNA in response to ArcN insulin. In conclusion, in females, ArcN insulin increases LSNA, in part, by suppressing tonic PVN NPY inhibition, which unmasks excitatory α-MSH drive of LSNA. Moreover, the steadily increasing rise in LSNA induced by ArcN insulin is also dependent on PVN MC3/4R.

Cerebrospinal Fluid Hypernatremia Elevates Sympathetic Nerve Activity and Blood Pressure via the Rostral Ventrolateral Medulla.

Elevated NaCl concentrations of the cerebrospinal fluid increase sympathetic nerve activity (SNA) in salt-sensitive hypertension. Neurons of the rostral ventrolateral medulla (RVLM) play a pivotal role in the regulation of SNA and receive mono- or polysynaptic inputs from several hypothalamic structures responsive to hypernatremia. Therefore, the present study investigated the contribution of RVLM neurons to the SNA and pressor response to cerebrospinal fluid hypernatremia. Lateral ventricle infusion of 0.15 mol/L, 0.6 mol/L, and 1.0 mol/L NaCl (5 µL/10 minutes) produced concentration-dependent increases in lumbar SNA, adrenal SNA, and arterial blood pressure, despite no change in splanchnic SNA and a decrease in renal SNA. Ganglionic blockade with chlorisondamine or acute lesion of the lamina terminalis blocked or significantly attenuated these responses, respectively. RVLM microinjection of the gamma-aminobutyric acid (GABAA) agonist muscimol abolished the sympathoexcitatory response to intracerebroventricular infusion of 1 mol/L NaCl. Furthermore, blockade of ionotropic glutamate, but not angiotensin II type 1, receptors significantly attenuated the increase in lumbar SNA, adrenal SNA, and arterial blood pressure. Finally, single-unit recordings of spinally projecting RVLM neurons revealed 3 distinct populations based on discharge responses to intracerebroventricular infusion of 1 mol/L NaCl: type I excited (46%; 11/24), type II inhibited (37%; 9/24), and type III no change (17%; 4/24). All neurons with slow conduction velocities were type I cells. Collectively, these findings suggest that acute increases in cerebrospinal fluid NaCl concentrations selectively activate a discrete population of RVLM neurons through glutamate receptor activation to increase SNA and arterial blood pressure.

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

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

Role of the Paraventricular Nucleus of the Hypothalamus in the Sympathoexcitatory Effects of Leptin.

Leptin binds to receptors in multiple hypothalamic nuclei to increase sympathetic nerve activity; however, the neurocircuitry is unclear. Here, using anesthetized male Sprague-Dawley rats, we investigated the role of the paraventricular nucleus of the hypothalamus. Intracerebroventricular injection of leptin slowly increased lumbar sympathetic nerve activity (LSNA), heart rate, mean arterial pressure, and baroreflex control of LSNA and heart rate. Inhibition of the paraventricular nucleus with muscimol completely reversed leptin's effects. Blockade of paraventricular melanocortin 3/4 receptors with SHU9119 or ionotropic glutamate receptors with kynurenate, alone or together, each partially reversed the effects of leptin, implicating increased activation of glutamate and melanocortin 3/4 receptors. Conversely, although blockade of neuropeptide Y Y1 receptors in the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate, these responses were prevented by intracerebroventricular or arcuate nucleus injections of leptin, suggesting that, at least in part, leptin also increases sympathetic nerve activity by suppression of tonic neuropeptide Y inhibitory inputs from the arcuate nucleus. Injection of the melanocortin 3/4 receptor agonist melanotan-II into the paraventricular nucleus increased LSNA, mean arterial pressure, and heart rate only after blockade of neuropeptide Y Y1 receptors. Therefore, we conclude that leptin increases LSNA in part via increased glutamatergic and α-melanocyte-stimulating hormone drive of paraventricular sympathoexcitatory neurons, the latter of which requires simultaneous withdrawal of tonic neuropeptide Y inhibition.

Salt-induced sympathoexcitation involves vasopressin V1a receptor activation in the paraventricular nucleus of the hypothalamus

A high-salt diet can lead to hydromineral imbalance and increases in plasma sodium and osmolality. It is recognized as one of the major contributing factors for cardiovascular diseases such as hypertension. The paraventricular nucleus (PVN) plays a pivotal role in osmotically driven sympathoexcitation and high blood pressure, the precise mechanisms of which are not fully understood. Recent evidence indicates that AVP released from magnocellular neurons might be involved in this process. Using a combination of in vivo and in situ studies, we sought to investigate whether AVP, acting on PVN neurons, can change mean arterial pressure (MAP) and sympathetic nerve activity (SNA) in euhydrated male rats. Furthermore, we wanted to determine whether V1a receptors on PVN neurons would be involved in salt-induced sympathoexcitation and hypertension. In rats, 4 days of salt loading (NaCl 2%) elicited a significant increase in plasma osmolality (39 ± 7 mosmol/kgH2O), an increase in MAP (26 ± 2 mmHg, P < 0.001), and sympathoexcitation compared with euhydrated rats. Microinjection of AVP into the PVN of conscious euhydrated animals (100 nl, 3 μM) elicited a pressor response (14 ± 2 mmHg) and a significant increase in lumbar SNA (100 nl, 1 mM) (19 ± 5%). Pretreatment with a V1a receptor antagonist, microinjected bilaterally into the PVN of salt-loaded animals, elicited a decrease in lumbar SNA (-14 ± 5%) and MAP (-19 ± 5 mmHg), when compared with the euhydrated group. Our findings show that AVP plays an important role in modulating the salt-induced sympathoexcitation and high blood pressure, via V1a receptors, within the PVN of male rats. As such, V1a receptors in the PVN might contribute to neurogenic hypertension in individuals consuming a high-salt diet.

PVN Neuropeptide Y Contributes to the Sympathoexcitatory Effect of Leptin

Intracerebroventricular (ICV) leptin administration increases sympathetic nerve activity (SNA) via a neuronal pathway that includes the paraventricular nucleus of the hypothalamus (PVN). PVN Neuropeptide Y (NPY) tonically inhibits SNA via type 1 receptors (Y1R). Leptin inhibits NPY neurons; however, whether leptin increases SNA in part by decreasing tonic PVN NPY inhibition is unknown. To test this hypothesis, we determined if blockade of PVN Y1R (Y1Rx) elicits smaller increases in lumbar SNA (LSNA) after ICV leptin (3 μg) in α‐chloralose anesthetized male SD rats. LSNA was increased 90 min after ICV leptin (to 158±8% of control, n=5; P&lt;0.05), but not after ICV artificial cerebrospinal fluid (aCSF; to 112±2%, n=5). In rats given ICV aCSF, subsequent PVN Y1Rx (bilateral BIBO3304, 60 pmol/60 nL) increased LSNA to 146±5 % of control (P&lt;0.05). However, after ICV leptin, neither PVN Y1Rx (to 171±10 %) nor PVN aCSF (160±18 to 177±19%, n=5) significantly increased LSNA. These data support our hypothesis that leptin reduces tonic PVN NPY inhibition of LSNA. Leptin increases SNA in part via activation of PVN melanocortin 3/4 receptors (MC3/4R), and PVN presympathetic neurons express both Y1R and MC4R. Therefore, to test if leptin‐induced decreases in PVN NPY inhibition unmask MC3/4R excitation, we next determined if the MC3/4R agonist MTII increases LSNA more after Y1Rx. Bilateral PVN MTII (60 pmol/60 nL) did not alter LSNA (110±5% to 112±10 %, n=3); however, after Y1Rx, MTII increased LSNA from 124±3% to 164±6% (P&lt;0.05, n=4). In conclusion, leptin decreases tonic PVN NPY inhibition of SNA, which may contribute to leptin‐induced increases in SNA by exposing enhanced MC3/4R excitatory drive of PVN presympathetic neurons. Supported by NIH HL088552 &amp; AHA.

Glutamate receptors in the hypothalamic paraventricular nucleus contribute to insulin-induced sympathoexcitation.

The sympathoexcitatory response to insulin is mediated by neurons in the arcuate nucleus (ARC) and hypothalamic paraventricular nucleus (PVH). Previous studies have reported that stimulation of ARC neurons increases sympathetic nerve activity (SNA) and arterial blood pressure (ABP) through glutamate receptor activation in the PVH. Therefore, the purpose of the present study was to determine whether glutamatergic neurotransmission in the PVH contributes to insulin-induced sympathoexcitation. Male Sprague-Dawley rats (275-400 g) were infused with isotonic saline or insulin (3.75 mU · kg(-1) · min(-1)) plus 50% dextrose to maintain euglycemia. Intravenous infusion of insulin significantly increased lumbar SNA without a significant change in mean ABP, renal SNA, heart rate, or blood glucose. Bilateral PVH injection of the excitatory amino acid antagonist kynurenic acid (KYN) lowered lumbar SNA and ABP of animals infused with insulin. Similarly, a cocktail of the NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced lumbar SNA and mean ABP during infusion of insulin. In a final experiment, bilateral PVH injection of AP5 only, but not CNQX, lowered lumbar SNA and mean ABP of animals infused with insulin. The peak changes in lumbar SNA and mean ABP of insulin-treated animals were not different between KYN, AP5 plus CNQX, or AP5 alone. These drug treatments did not alter any variable in animals infused with saline. Altogether, these findings suggest that glutamatergic NMDA neurotransmission in the PVH contributes to insulin-induced sympathoexcitation.