Functional knockout of the TRPV1 channel has no effect on the exercise pressor reflex in rats.

The role played by the transient receptor potential vanilloid 1 (TRPV1) channel on the thin fibre afferents evoking the exercise pressor reflex is controversial. To shed light on this controversy, we compared the exercise pressor reflex between newly developed TRPV1+/+ , TRPV1+/- and TRPV1-/- rats. Carotid arterial injection of capsaicin (0.5 µg), evoked significant pressor responses in TRPV1+/+ and TRPV1+/- rats, but not in TRPV1-/- rats. In acutely isolated dorsal root ganglion neurons innervating the gastrocnemius muscles, capsaicin evoked inward currents in neurons isolated from TRPV1+/+ and TRPV1+/- rats but not in neurons isolated from TRPV1-/- rats. The reflex was evoked by stimulating the tibial nerve in decerebrated rats whose femoral artery was either freely perfused or occluded. We found no difference between the reflex in the three groups of rats regardless of the patency of the femoral artery. For example, the peak pressor responses to contraction in TRPV1+/+ , TRPV1+/- and TRPV1-/- rats with patent femoral arteries averaged 17.1 ± 7.2, 18.9 ± 12.4 and 18.4 ± 8.6 mmHg, respectively. Stimulation of the tibial nerve after paralysis with pancuronium had no effect on arterial pressure, findings which indicated that the pressor responses to contraction were not caused by electrical stimulation of afferent tibial nerve axons. We also found that expression levels of acid-sensing ion channel 1 and endoperoxide 4 receptor in the L4 and 5 dorsal root ganglia were not upregulated in the TRPV1-/- rats. We conclude that TRPV1 is not needed to evoke the exercise pressor reflex in rats whose contracting muscles have either a patent or an occluded arterial blood supply. KEY POINTS: A reflex arising in contracting skeletal muscle contributes to the increases in arterial blood pressure, cardiac output and breathing evoked by exercise. The sensory arm of the reflex comprises both mechanoreceptors and metaboreceptors, of which the latter signals that blood flow to exercising muscle is not meeting its metabolic demand. The nature of the channel on the metaboreceptor sensing a mismatch between supply and demand is controversial; some believe that it is the transient receptor potential vanilloid 1 (TRPV1) channel. Using genetically engineered rats in which the TRPV1 channel is rendered non-functional, we have shown that it is not needed to evoke the metaboreflex.

Sympathoexcitatory responses to renal chemosensitive stimuli are exaggerated at nighttime in rats.

The circadian cycle impacts sympathetic nerve activity (SNA), cardiovascular hemodynamics, and renal function. Activation of renal sensory nerves by chemosensory and mechanosensory stimuli reflexively changes efferent SNA and arterial blood pressure (ABP) to maintain homeostasis. However, it is unclear to what extent circadian cycle influences reflex SNA and ABP responses to renal sensory stimuli. Renal, splanchnic, and lumbar SNA and ABP responses to intrarenal arterial infusion of bradykinin or capsaicin and elevated renal pelvic pressure were measured in male and female Sprague-Dawley rats during nighttime (wakeful/active phase) and daytime (inactive phase). Intrarenal arterial bradykinin infusion significantly increased efferent renal SNA, splanchnic SNA, and ABP but not lumbar SNA. Responses were greater during nighttime versus daytime. Similarly, intrarenal arterial capsaicin infusion significantly increased renal SNA and splanchnic SNA, and responses were again greater during nighttime. Elevated renal pelvic pressure increased renal SNA and splanchnic SNA; however, responses did not differ between daytime and nighttime. Finally, afferent renal nerve activity responses to bradykinin were not different between daytime and nighttime. Thus, renal chemokines elicit greater sympathoexcitatory responses at nighttime that cannot be attributed to differences in afferent renal nerve activity. Collectively, these data suggest that the circadian cycle alters the excitability of central autonomic networks to alter baseline SNA and ABP as well as the magnitude of visceral reflexes.NEW & NOTEWORTHY The current study discovers that the circadian cycle influences sympathetic and hemodynamic responses to activation of renal chemosensitive sensory fibers. Sympathetic responses to intrarenal bradykinin or capsaicin infusion were exaggerated during nighttime (active period), but mechanosensitive responses to elevated renal pelvic pressure were not. Importantly, renal afferent nerve responses were not different between nighttime and daytime. These data suggest that the circadian cycle modulates sympathetic responses to visceral afferent activation.

Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Impact of anesthesia and sex on sympathetic efferent and hemodynamic responses to renal chemo- and mechanosensitive stimuli.

Activation of renal sensory nerves by chemo- and mechanosensitive stimuli produces changes in efferent sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Anesthesia and sex influence autonomic function and cardiovascular hemodynamics, but it is unclear to what extent anesthesia and sex impact SNA and ABP responses to renal sensory stimuli. We measured renal, splanchnic, and lumbar SNA and ABP in male and female Sprague-Dawley rats during contralateral renal infusion of capsaicin and bradykinin or during elevation in renal pelvic pressure. Responses were evaluated with a decerebrate preparation or Inactin, urethane, or isoflurane anesthesia. Intrarenal arterial infusion of capsaicin (0.1-30.0 µM) increased renal SNA, splanchnic SNA, or ABP but decreased lumbar SNA in the Inactin group. Intrarenal arterial infusion of bradykinin (0.1-30.0 µM) increased renal SNA, splanchnic SNA, and ABP but decreased lumbar SNA in the Inactin group. Elevated renal pelvic pressure (0-20 mmHg, 30 s) significantly increased renal SNA and splanchnic SNA but not lumbar SNA in the Inactin group. In marked contrast, SNA and ABP responses to every renal stimulus were severely blunted in the urethane and decerebrate groups and absent in the isoflurane group. In the Inactin group, the magnitude of SNA responses to chemo- and mechanosensory stimuli were not different between male and female rats. Thus, chemo- and mechanosensitive stimuli produce differential changes in renal, splanchnic, and lumbar SNA. Experimentally, future investigations should consider Inactin anesthesia to examine sympathetic and hemodynamic responses to renal sensory stimuli.NEW & NOTEWORTHY The findings highlight the impact of anesthesia, and to a lesser extent sex, on sympathetic efferent and hemodynamic responses to chemosensory and mechanosensory renal stimuli. Sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses were present in Inactin-anesthetized rats but largely absent in decerebrate, isoflurane, or urethane preparations. Renal chemosensory stimuli differentially changed SNA: renal and splanchnic SNA increased, but lumbar SNA decreased. Future investigations should consider Inactin anesthesia to study SNA and hemodynamic responses to renal sensory nerve activation.

Genetic or pharmacologic Nrf2 activation increases proteinuria in chronic kidney disease in mice.

The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway upregulates key cellular defenses. Clinical trials are utilizing pharmacologic Nrf2 inducers such as bardoxolone methyl to treat chronic kidney disease, but Nrf2 activation has been linked to a paradoxical increase in proteinuria. To understand this effect, we examined genetically engineered mice with elevated Nrf2 signaling due to reduced expression of the Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1). These Keap1FA/FA mice lacked baseline proteinuria but exhibited increased proteinuria in experimental models evoked by adriamycin, angiotensin II, or protein overload. After injury, Keap1FA/FA mice had increased glomerulosclerosis, nephrin disruption and shedding, podocyte injury, foot process effacement, and interstitial fibrosis. Keap1FA/FA mice also had higher daytime blood pressures and lower heart rates measured by radiotelemetry. Conversely, Nrf2 knockout mice were protected from proteinuria. We also examined the pharmacologic Nrf2 inducer CDDO-Im. Compared to angiotensin II alone, the combination of angiotensin II and CDDO-Im significantly increased proteinuria, a phenomenon not observed in Nrf2 knockout mice. This effect was not accompanied by additional increases in blood pressure. Finally, Nrf2 was found to be upregulated in the glomeruli of patients with focal segmental glomerulosclerosis, diabetic nephropathy, fibrillary glomerulonephritis, and membranous nephropathy. Thus, our studies demonstrate that Nrf2 induction in mice may exacerbate proteinuria in chronic kidney disease.

Impact of anesthesia, sex, and circadian cycle on renal afferent nerve sensitivity.

Elevated renal afferent nerve (ARNA) activity or dysfunctional reno-renal reflexes via altered ARNA sensitivity contribute to hypertension and chronic kidney disease. These nerves contain mechano- and chemosensitive fibers that respond to ischemia, changes in intrarenal pressures, and chemokines. Most studies have utilized various anesthetized preparations and exclusively male animals to characterize ARNA responses. Therefore, this study assessed the impact of anesthesia, sex, and circadian period on ARNA responses and sensitivity. Multifiber ARNA recordings were performed in male and female Sprague-Dawley rats (250-400 g) and compared across decerebrate versus Inactin, isoflurane, and urethane anesthesia groups. Intrarenal artery infusion of capsaicin (0.1-50.0 µM, 0.05 mL) produced concentration-dependent increases in ARNA; however, the ARNA sensitivity was significantly greater in decerebrate versus Inactin, isoflurane, and urethane groups. Increases in renal pelvic pressure (0-30 mmHg, 30 s) produced pressure-dependent increases in ARNA; however, ARNA sensitivity was again greater in decerebrate and Inactin groups versus isoflurane and urethane. Acute renal artery occlusion (30 s) increased ARNA, but responses did not differ across groups. Analysis of ARNA responses to increased pelvic pressure between male and female rats revealed significant sex differences only in isoflurane and urethane groups. ARNA responses to intrarenal capsaicin infusion were significantly blunted at nighttime versus daytime; however, ARNA responses to increased pelvic pressure or renal artery occlusion were not different between daytime and nighttime. These results demonstrate that ARNA sensitivity is greatest in decerebrate and Inactin-anesthetized groups but was not consistently influenced by sex.NEW & NOTEWORTHY We determined the impact of anesthesia, sex, and circadian cycle on renal afferent nerve (ARNA) sensitivity to chemical and mechanical stimuli. ARNA sensitivity to renal capsaicin infusion was greatest in decerebrate > Inactin > urethane or isoflurane groups. Elevated renal pelvic pressure significantly increased ARNA; decerebrate and Inactin groups exhibited the greatest ARNA sensitivity. Sex differences in renal afferent responses were not consistently observed. Circadian cycle altered chemosensory but not mechanosensory responses.

Ultrasound super-resolution imaging provides a noninvasive assessment of renal microvasculature changes during mouse acute kidney injury.

Acute kidney injury (AKI) is a risk factor for the development of chronic kidney disease (CKD). One mechanism for this phenomenon is renal microvascular rarefaction and subsequent chronic impairment in perfusion. However, diagnostic tools to monitor the renal microvasculature in a noninvasive and quantitative manner are still lacking. Ultrasound super-resolution imaging is an emerging technology that can identify microvessels with unprecedented resolution. Here, we applied this imaging technique to identify microvessels in the unilateral ischemia-reperfusion injury mouse model of AKI-to-CKD progression in vivo. Kidneys from 21 and 42 day post- ischemia-reperfusion injury, the contralateral uninjured kidneys, and kidneys from sham-operated mice were examined by ultrasound super-resolution and histology. Renal microvessels were successfully identified by this imaging modality with a resolution down to 32 µm. Renal fibrosis was observed in all kidneys with ischemia-reperfusion injury and was associated with a significant reduction in kidney size, cortical thickness, relative blood volume, and microvascular density as assessed by this imaging. Tortuosity of the cortical microvasculature was also significantly increased at 42 days compared to sham. These vessel density measurements correlated significantly with CD31 immunohistochemistry (R2=0.77). Thus, ultrasound super-resolution imaging provides unprecedented resolution and is capable of noninvasive quantification of renal vasculature changes associated with AKI-to-CKD progression in mice. Hence, this technique could be a promising diagnostic tool for monitoring progressive kidney disease.

Excessive dietary salt promotes aortic stiffness in murine renovascular hypertension.

Renovascular hypertension is characterized by activation of the renin-angiotensin-aldosterone system, blunted natriuretic responses, and elevated sympathetic nerve activity. Excess dietary salt intake exaggerates arterial blood pressure (ABP) in multiple models of experimental hypertension. The present study tested whether a high-salt diet exaggerated ABP and vascular dysfunction in a 2-kidney, 1-clip (2K1C) murine model. Male C57BL/6J mice (8-12 wk) were randomly assigned, and fed a 0.1% or 4.0% NaCl diet, and instrumented with telemetry units to measure ABP. Then, the 2K1C model was produced by placing a cuff around the right renal artery. Systolic, diastolic, and mean ABP were significantly higher in mice fed 4.0% vs. 0.1% NaCl at 1 wk but not after 3 wk. Interestingly, 2K1C hypertension progressively increased arterial pulse pressure in both groups; however, the magnitude was significantly greater in mice fed 4.0% vs. 0.1% NaCl at 3 wk. Moreover, pulse wave velocity was significantly greater in 2K1C mice fed 4.0% vs. 0.1% NaCl diet or sham-operated mice fed either diet. Histological assessment of aortas indicated no structural differences among groups. Finally, endothelium-dependent vasodilation was significantly and selectively attenuated in the aorta but not mesenteric arteries of 2K1C mice fed 4.0% NaCl vs. 0.1% NaCl or sham-operated control mice. The findings suggest that dietary salt loading transiently exaggerates 2K1C renovascular hypertension but promotes chronic aortic stiffness and selective aortic vascular dysfunction.NEW & NOTEWORTHY High dietary salt exaggerates hypertension in multiple experimental models. Here we demonstrate that a high-salt diet produces a greater increase in arterial blood pressure at 1 wk after induction of 2-kidney, 1-clip (2K1C) hypertension but not at 3 wk. Interestingly, 2K1C mice fed a high-salt diet displayed an exaggerated pulse pressure, elevated pulse wave velocity, and reduced endothelium-dependent vasodilation of the aorta but not mesenteric arteries. These findings suggest that dietary salt may interact with underlying cardiovascular disease to promote selective vascular dysfunction and aortic stiffness.

Short-term water deprivation does not increase blood pressure variability or impair neurovascular function in healthy young adults.

High dietary salt increases arterial blood pressure variability (BPV) in salt-resistant, normotensive rodents and is thought to result from elevated plasma [Na+] sensitizing central sympathetic networks. Our purpose was to test the hypothesis that water deprivation (WD)-induced elevations in serum [Na+] augment BPV via changes in baroreflex function and sympathetic vascular transduction in humans. In a randomized crossover fashion, 35 adults [17 female/18 male, age: 25 ± 4 yr, systolic/diastolic blood pressure (BP): 107 ± 11/60 ± 7 mmHg, body mass index: 23 ± 3 kg/m2] completed two hydration protocols: a euhydration control condition (CON) and a stepwise reduction in water intake over 3 days, concluding with 16 h of WD. We assessed blood and urine electrolyte concentrations and osmolality, resting muscle sympathetic nerve activity (MSNA; peroneal microneurography; 18 paired recordings), beat-to-beat BP (photoplethysmography), common femoral artery blood flow (Doppler ultrasound), and heart rate (single-lead ECG). A subset of participants (n = 25) underwent ambulatory BP monitoring during day 3 of each protocol. We calculated average real variability as an index of BPV. WD increased serum [Na+] (141.0 ± 2.3 vs. 142.1 ± 1.7 mmol/L, P < 0.01) and plasma osmolality (288 ± 4 vs. 292 ± 5 mosmol/kg H2O, P < 0.01). However, WD did not increase beat-to-beat (1.9 ± 0.4 vs. 1.8 ± 0.4 mmHg, P = 0.24) or ambulatory daytime (9.6 ± 2.1 vs. 9.4 ± 3.3 mmHg, P = 0.76) systolic BPV. Additionally, sympathetic baroreflex sensitivity (P = 0.20) and sympathetic vascular transduction were not different after WD (P = 0.17 for peak Δmean BP following spontaneous MSNA bursts). These findings suggest that, despite modestly increasing serum [Na+], WD does not affect BPV, arterial baroreflex function, or sympathetic vascular transduction in healthy young adults.

Missing pieces of the Piezo1/Piezo2 baroreceptor hypothesis: an autonomic perspective.

Baroreceptors play a pivotal role in the regulation of blood pressure through moment to moment sensing of arterial blood pressure and providing information to the central nervous system to make autonomic adjustments to maintain appropriate tissue perfusion. A recent publication by Zeng and colleagues (Zeng WZ, Marshall KL, Min S, Daou I, Chapleau MW, Abboud FM, Liberles SD, Science 362: 464-467, 2018) suggests the mechanosensitive ion channels Piezo1 and Piezo2 represent the cellular mechanism by which baroreceptor nerve endings sense changes in arterial blood pressure. However, before Piezo1 and Piezo2 are accepted as the sensor of baroreceptors, the question must be asked of what criteria are necessary to establish this and how well the report of Zeng and colleagues (Zeng WZ, Marshall KL, Min S, Daou I, Chapleau MW, Abboud FM, Liberles SD, Science 362: 464-467, 2018) satisfies these criteria. We briefly review baroreceptor function, outline criteria that a putative neuronal sensor of blood pressure must satisfy, and discuss whether the recent findings of Zeng and colleagues suitably meet these criteria. Despite the provocative hypothesis, there are significant concerns regarding the evidence supporting a role of Piezo1/Piezo2 in arterial baroreceptor function.

Renal sensory nerves increase sympathetic nerve activity and blood pressure in 2-kidney 1-clip hypertensive mice.

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to define the function, anatomy, and contribution of mouse renal sensory neurons to a renal nerve-dependent model of hypertension. First, electrical stimulation of mouse renal afferent nerves produced frequency-dependent increases in ABP that were eliminated by ganglionic blockade. Stimulus-triggered averaging revealed renal afferent stimulation significantly increased splanchnic, renal, and lumbar sympathetic nerve activity (SNA). Second, kidney injection of wheat germ agglutinin into male C57Bl6 mice (12-14 wk; Jackson Laboratories) produced ipsilateral labeling in the T11-L2 dorsal root ganglia. Next, 2-kidney 1-clip (2K1C) hypertension was produced in male C57Bl6 mice (12-14 wk; Jackson Laboratories) by placement of a 0.5-mm length of polytetrafluoroethylene tubing around the left renal artery. 2K1C mice displayed an elevated ABP measured via telemetry and a greater fall in mean ABP to ganglionic blockade at day 14 or 21 vs. day 0. Renal afferent discharge was significantly higher in 2K1C-clipped vs. 2K1C-unclipped or sham kidneys. In addition, 2K1C-clipped vs. 2K1C-unclipped or sham kidneys had lower renal mass and higher mRNA levels of several proinflammatory cytokines. Finally, both ipsilateral renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33 mM capsaicin) at time of clipping resulted in lower ABP of 2K1C mice at day 14 or 21. These findings suggest mouse renal sensory neurons are activated to increase SNA and ABP in 2K1C hypertension. NEW & NOTEWORTHY This study documents the function, anatomy, and contribution of mouse renal sensory nerves to neurogenic hypertension produced by renal stenosis. Activation of renal afferents increased sympathetic nerve activity and blood pressure. Renal afferent activity was elevated in hypertensive mice, and renal afferent denervation lowered blood pressure. Clinically, patients with renal stenosis have been excluded from clinical trials for renal denervation, but this study highlights the potential therapeutic efficacy to target renal nerves in these patients.

The influence of acute elevations in plasma osmolality and serum sodium on sympathetic outflow and blood pressure responses to exercise.

Elevated plasma osmolality (pOsm) has been shown to increase resting sympathetic nerve activity in animals and humans. The present study tested the hypothesis that increases in pOsm and serum sodium (sNa+) concentration would exaggerate muscle sympathetic nerve activity (MSNA) and blood pressure (BP) responses to handgrip (HG) exercise and postexercise ischemia (PEI). BP and MSNA were measured during HG followed by PEI before and after a 23-min hypertonic saline infusion (HSI-3% NaCl). Eighteen participants (age 23 ± 1 yr; BMI 24 ± 1 kg/m2) completed the protocol; pOsm and sNa+ increased from pre- to post-HSI (285 ± 1 to 291 ± 1 mosmol/kg H2O; 138.2 ± 0.3 to 141.3 ± 0.4 mM; P < 0.05 for both). Resting mean BP (90 ± 2 vs. 92 ± 1 mmHg) and MSNA (11 ± 2 vs. 15 ± 2 bursts/min) were increased pre- to post-HSI ( P < 0.05 for both). Mean BP responses to HG (106 ± 2 vs. 111 ± 2 mmHg, P < 0.05) and PEI (102 ± 2 vs. 107 ± 2 mmHg, P < 0.05) were higher post-HSI. Similarly, MSNA during HG (20 ± 2 vs. 29 ± 2 bursts/min, P < 0.05) and PEI (19 ± 2 vs. 24 ± 3 bursts/min, P < 0.05) were greater post-HSI. In addition, the change in MSNA was greater post-HSI during HG (Δ9 ± 2 vs. Δ13 ± 3 bursts/min, P < 0.05). A second set of participants ( n = 13, age 23 ± 1 yr; BMI 24 ± 1 kg/m2) completed a time control (TC) protocol consisting of quiet rest instead of an infusion. The TC condition yielded no change in resting sNa+, pOsm, mean BP, or MSNA (all P > 0.05); responses to HG and PEI were not different pre- to post-quiet rest ( P > 0.05). In summary, acutely increasing pOsm and sNa+ exaggerates BP and MSNA responses during HG exercise and PEI. NEW & NOTEWORTHY Elevated plasma osmolality has been shown to increase resting sympathetic activity and blood pressure. This study provides evidence that acute elevations in plasma osmolality and serum sodium exaggerated muscle sympathetic nerve activity and blood pressure responses during exercise pressor reflex activation in healthy young adults.

Alterations in dietary sodium intake affect cardiovagal baroreflex sensitivity.

High dietary sodium intake has been linked to alterations in neurally mediated cardiovascular function, but the effects of high sodium on cardiovagal baroreflex sensitivity (cBRS) in healthy adults are unknown. The purpose of this study was to determine whether high dietary sodium alters cBRS and heart rate variability (HRV) and whether acute intravenous sodium loading similarly alters cBRS and HRV. High dietary sodium (300 mmol/day, 7 days) was compared with low dietary sodium (20 mmol/day, 7 days; randomized) in 14 participants (38 ± 4 yr old, 23 ± 1 kg/m2 body mass index, 7 women). Acute sodium loading was achieved via a 23-min intravenous hypertonic saline infusion (HSI) in 14 participants (22 ± 1 yr old, 23 ± 1 kg/m2 body mass index, 7 women). During both protocols, participants were supine for 5 min during measurement of beat-to-beat blood pressure (photoplethysmography) and R-R interval (ECG). cBRS was evaluated using the sequence method. Root mean square of successive differences in R-R interval (RMSSD) was used as an index of HRV. Serum sodium (137.4 ± 0.7 vs. 139.9 ± 0.5 meq/l, P < 0.05), plasma osmolality (285 ± 1 vs. 289 ± 1 mosmol/kgH2O, P < 0.05), cBRS (18 ± 2 vs. 26 ± 3 ms/mmHg, P < 0.05), and RMSSD (62 ± 6 vs. 79 ± 10 ms, P < 0.05) were increased following high-sodium diet intake compared with low-sodium diet intake. HSI increased serum sodium (138.1 ± 0.4 vs. 141.1 ± 0.5 meq/l, P < 0.05) and plasma osmolality (286 ± 1 vs. 290 ± 1 mosmol/kgH2O, P < 0.05) but did not change cBRS (26 ± 5 vs. 25 ± 3 ms/mmHg, P = 0.73) and RMSSD (63 ± 9 vs. 63 ± 8 ms, P = 0.99). These data suggest that alterations in dietary sodium intake alter cBRS and HRV but that acute intravenous sodium loading does not alter these indexes of autonomic cardiovascular regulation.

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

KEY POINTS: 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. ABSTRACT: 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 (5 µl/10 min) of 1.0 m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0 osmol l-1 ). Second, OVLT microinjection (20 nl) of 1.0 m 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 (+7.5 mm) and mannitol (+15 mm). 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 0.5 m 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.

Hypothalamic Signaling in Body Fluid Homeostasis and Hypertension.

PURPOSE OF REVIEW: The central nervous system plays a pivotal role in the regulation of extracellular fluid volume and consequently arterial blood pressure. Key hypothalamic regions sense and integrate neurohumoral signals to subsequently alter intake (thirst and salt appetite) and output (renal excretion via neuroendocrine and autonomic function). Here, we review recent findings that provide new insight into such mechanisms that may represent new therapeutic targets. RECENT FINDINGS: Implementation of cutting edge neuroscience approaches such as opto- and chemogenetics highlight pivotal roles of circumventricular organs to impact body fluid homeostasis. Key signaling mechanisms within these areas include the N-terminal variant of transient receptor potential vannilloid type-1, NaX, epithelial sodium channel, brain electroneutral transporters, and non-classical actions of vasopressin. Despite the identification of several new mechanisms, future studies need to better define the neurochemical phenotype and molecular profiles of neurons within circumventricular organs for future therapeutic potential.

High fat diet attenuates glucose-dependent facilitation of 5-HT3 -mediated responses in rat gastric vagal afferents.

KEY POINTS: Glucose regulates the density and function of 5-HT3 receptors on gastric vagal afferent neurones. Diet-induced obesity compromises the excitability and responsiveness of vagal afferents. In this study, we assessed whether exposure to a high fat diet (HFD) compromises the glucose-dependent modulation of 5-HT responses in gastric vagal afferents prior to the development of obesity. We show that HFD does not alter the response of gastric vagal afferent nerves and neurones to 5-HT but attenuates the ability of glucose to amplify 5-HT3-induced responses. These results suggest that glucose-dependent vagal afferent signalling is compromised by relatively short periods of exposure to HFD well in advance of the development of obesity or glycaemic dysregulation. Glucose regulates the density and function of 5-HT3 receptors on gastric vagal afferent neurones. Since diet-induced obesity attenuates the responsiveness of gastric vagal afferents to several neurohormones, the aim of the present study was to determine whether high fat diet (HFD) compromises the glucose-dependent modulation of 5-HT responses in gastric vagal afferents prior to the development of obesity. Rats were fed control or HFD (14% or 60% kilocalories from fat, respectively) for up to 8 weeks. Neurophysiological recordings assessed the ability of 5-HT to increase anterior gastric vagal afferent nerve (VAN) activity in vivo before and after acute hyperglycaemia, while electrophysiological recordings from gastric-projecting nodose neurones assessed the ability of glucose to modulate the 5-HT response in vitro. Immunocytochemical studies determined alterations in the neuronal distribution of 5-HT3 receptors. 5-HT and cholecystokinin (CCK) induced dose-dependent increases in VAN activity in all rats; HFD attenuated the response to CCK, but not 5-HT. The 5-HT-induced response was amplified by acute hyperglycaemia in control, but not HFD, rats. Similarly, although 5-HT induced an inward current in both control and HFD gastric nodose neurones in vitro, the 5-HT response and receptor distribution was amplified by acute hyperglycaemia only in control rats. These data suggest that, while HFD does not affect the response of gastric-projecting vagal afferents to 5-HT, it attenuates the ability of glucose to amplify 5-HT effects. This suggests that glucose-dependent vagal afferent signalling is compromised by short periods of exposure to HFD well in advance of obesity or glycaemic dysregulation.

DREADD-induced activation of subfornical organ neurons stimulates thirst and salt appetite.

The subfornical organ (SFO) plays a pivotal role in body fluid homeostasis through its ability to integrate neurohumoral signals and subsequently alter behavior, neuroendocrine function, and autonomic outflow. The purpose of the present study was to evaluate whether selective activation of SFO neurons using virally mediated expression of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) stimulated thirst and salt appetite. Male C57BL/6 mice (12-15 wk) received an injection of rAAV2-CaMKII-HA-hM3D(Gq)-IRES-mCitrine targeted at the SFO. Two weeks later, acute injection of clozapine N-oxide (CNO) produced dose-dependent increases in water intake of mice with DREADD expression in the SFO. CNO also stimulated the ingestion of 0.3 M NaCl. Acute injection of CNO significantly increased the number of Fos-positive nuclei in the SFO of mice with robust DREADD expression. Furthermore, in vivo single-unit recordings demonstrate that CNO significantly increases the discharge frequency of both ANG II- and NaCl-responsive neurons. In vitro current-clamp recordings confirm that bath application of CNO produces a significant membrane depolarization and increase in action potential frequency. In a final set of experiments, chronic administration of CNO approximately doubled 24-h water intake without an effect on salt appetite. These findings demonstrate that DREADD-induced activation of SFO neurons stimulates thirst and that DREADDs are a useful tool to acutely or chronically manipulate neuronal circuits influencing body fluid homeostasis.

Hypernatremia-induced vasopressin secretion is not altered in TRPV1-/- rats.

Changes in osmolality or extracellular NaCl concentrations are detected by specialized neurons in the hypothalamus to increase vasopressin (VP) and stimulate thirst. Recent in vitro evidence suggests this process is mediated by an NH2-terminal variant of the transient receptor potential vanilloid type 1 (TRPV1) channel expressed by osmosensitive neurons of the lamina terminalis and vasopressinergic neurons of the supraoptic nucleus. The present study tested this hypothesis in vivo by analysis of plasma VP levels during acute hypernatremia in awake control and TRPV1(-/-) rats. TRPV1(-/-) rats were produced by a Zinc-finger-nuclease 2-bp deletion in exon 13. Intravenous injection of the TRPV1 agonist capsaicin produced hypotension and bradycardia in control rats, but this response was absent in TRPV1(-/-) rats. Infusion of 2 M NaCl (1 ml/h iv) increased plasma osmolality, electrolytes, and VP levels in both control and TRPV1(-/-) rats. However, plasma VP levels did not differ between strains at any time. Furthermore, a linear regression between plasma VP versus osmolality revealed a significant correlation in both control and TRPV1(-/-) rats, but the slope of the regression lines was not attenuated in TRPV1(-/-) versus control rats. Hypotension produced by intravenous injection of minoxidil decreased blood pressure and increased plasma VP levels similarly in both groups. Finally, both treatments stimulated thirst; however, cumulative water intakes in response to hypernatremia or hypotension were not different between control and TRPV1(-/-) rats. These findings suggest that TRPV1 channels are not necessary for VP secretion and thirst stimulated by hypernatremia.

Peripheral venous distension elicits a blood pressure raising reflex in young and middle-aged adults.

Distension of peripheral veins in humans elicits a pressor and sympathoexcitatory response that is mediated through group III/IV skeletal muscle afferents. There is some evidence that autonomic reflexes mediated by these sensory fibers are blunted with increasing age, yet to date the venous distension reflex has only been studied in young adults. Therefore, we tested the hypothesis that the venous distension reflex would be attenuated in middle-aged compared with young adults. Nineteen young (14 men/5 women, 25 ± 1 yr) and 13 middle-aged (9 men/4 women, 50 ± 2 yr) healthy normotensive participants underwent venous distension via saline infusion through a retrograde intravenous catheter in an antecubital vein during limb occlusion. Beat-by-beat blood pressure, muscle sympathetic nerve activity (MSNA), and model flow-derived cardiac output (Q), and total peripheral resistance (TPR) were recorded throughout the trial. Mean arterial pressure (MAP) increased during the venous distension in both young (baseline 83 ± 2, peak 94 ± 3 mmHg; P < 0.05) and middle-aged adults (baseline 88 ± 2, peak 103 ± 3 mmHg; P < 0.05). MSNA also increased in both groups [young: baseline 886 ± 143, peak 1,961 ± 242 arbitrary units (AU)/min; middle-aged: baseline 1,164 ± 225, peak 2,515 ± 404 AU/min; both P < 0.05]. TPR (P < 0.001), but not Q (P = 0.76), increased during the trial. However, the observed increases in blood pressure, MSNA, and TPR were similar between young and middle-aged adults. Additionally, no correlation was found between age and the response to venous distension (all P > 0.05). These findings suggest that peripheral venous distension elicits a pressor and sympathetic response in middle-aged adults similar to the response observed in young adults.

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.