Role of NaV1.7 in postganglionic sympathetic nerve function in human and guinea-pig arteries.

NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF 05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P < 0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA 121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF 05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.

Postganglionic sympathetic neurons, but not locus coeruleus optostimulation, activates neuromuscular transmission in the adult mouse in vivo.

Recent work demonstrated that sympathetic neurons innervate the skeletal muscle near the neuromuscular junction (NMJ), and muscle sympathectomy and sympathomimetic agents strongly influence motoneuron synaptic vesicle release ex vivo. Moreover, reports attest that the pontine nucleus locus coeruleus (LC) projects to preganglionic sympathetic neurons and regulates human mobility and skeletal muscle physiology. Thus, we hypothesized that peripheral and central sympathetic neurons projecting directly or indirectly to the skeletal muscle regulate NMJ transmission. The aim of this study was to define the specific neuronal groups in the peripheral and central nervous systems that account for such regulation in adult mice in vivo by using optogenetics and NMJ transmission recordings in 3-5-month-old, male and female ChR2(H134R/EYFP)/TH-Cre mice. After detecting ChR2(H134R)/EYFP fluorescence in the paravertebral ganglia and LC neurons, we tested whether optostimulating the plantar nerve near the lumbricalis muscle or LC neurons effectively modulates motor nerve terminal synaptic vesicle release in living mice. Nerve optostimulation increased motor synaptic vesicle release in vitro and in vivo, while the presynaptic adrenoceptor blockers propranolol (ß1/ß2) and atenolol (ß1) prevented this outcome. The effect is primarily presynaptic since miniature end-plate potential (MEPP) kinetics remained statistically unmodified after stimulation. In contrast, optostimulation of LC neurons did not regulate NMJ transmission. In summary, we conclude that postganglionic sympathetic neurons, but not LC neurons, increased NMJ transmission by acting on presynaptic ß1-adrenergic receptors in vivo.

Human epicardium-derived cells reinforce cardiac sympathetic innervation.

RATIONALE: After cardiac damage, excessive neurite outgrowth (sympathetic hyperinnervation) can occur, which is related to ventricular arrhythmias/sudden cardiac death. Post-damage reactivation of epicardium causes epicardium-derived cells (EPDCs) to acquire a mesenchymal character, contributing to cardiac regeneration. Whether EPDCs also contribute to cardiac re/hyperinnervation, is unknown. AIM: To investigate whether mesenchymal EPDCs influence cardiac sympathetic innervation. METHODS AND RESULTS: Sympathetic ganglia were co-cultured with mesenchymal EPDCs and/or myocardium, and neurite outgrowth and sprouting density were assessed. Results showed a significant increase in neurite density and directional (i.e. towards myocardium) outgrowth when ganglia were co-cultured with a combination of EPDCs and myocardium, as compared to cultures with EPDCs or myocardium alone. In absence of myocardium, this outgrowth was not directional. Neurite differentiation of PC12 cells in conditioned medium confirmed these results via a paracrine effect, in accordance with expression of neurotrophic factors in myocardial explants co-cultured with EPDCs. Of interest, EPDCs increased the expression of nerve growth factor (NGF) in cultured, but not in fresh myocardium, possibly due to an "ischemic state" of cultured myocardium, supported by TUNEL and Hif1α expression. Cardiac tissues after myocardial infarction showed robust NGF expression in the infarcted, but not remote area. CONCLUSION: Neurite outgrowth and density increases significantly in the presence of EPDCs by a paracrine effect, indicating a new role for EPDCs in the occurrence of sympathetic re/hyperinnervation after cardiac damage.

Sesame lignans increase sympathetic nerve activity and blood flow in rat skeletal muscles.

Beneficial effects of sesame lignans, especially antioxidative effects, have been widely reported; however, its potential effects on autonomic nerves have not yet been investigated. Therefore, the current study aimed to investigate the effect of sesame lignans on the autonomic nervous system. The sympathetic nerve activity in rat skeletal muscle was measured using electrophysiological approaches, with blood flow determined using the laser Doppler method. Sesame lignans were administered intragastrically at 2 and 20 mg/kg, and after 60 min, the sympathetic nerve activity was observed to increase by 45.2% and 66.1%, respectively. A significant increase in blood flow (39.6%) was also observed for the 20-mg/kg dose when measured at 55 min after administration. These sympathomimetic effects were completely prevented by subdiaphragmatic vagotomy, and the increase in blood flow was eliminated in the presence of the beta2-adrenergic receptor inhibitor butoxamine. Thus, it is proposed that sesame lignans can increase the blood flow of skeletal muscle, possibly by exciting sympathetic nerve activity through the afferent vagal nerve.

Elimination of spurious absent sweat response in QSWEAT recordings.

INTRODUCTION: Forearm QSWEAT recordings are occasionally absent in females, likely due to high skin resistance. METHODS: We identified consecutive subjects with no sudomotor abnormalities but absent/markedly reduced QSWEAT forearm volume, and repeated QSWEAT at the same site after gentle abrasion. RESULTS: QSWEAT volumes were absent for 4 subjects and markedly reduced for the other 4 (median 0.01, IQR 0-0.03). After gentle skin abrasion, repeat volumes were significantly higher for all subjects and became normal in 7 of 8 subjects. DISCUSSION: Skin abrasion restores QSWEAT volumes in previously absent/markedly reduced site suggesting that skin preparation using abrasion is more effective.

Dramatically Amplified Thoracic Sympathetic Postganglionic Excitability and Integrative Capacity Revealed with Whole-Cell Patch-Clamp Recordings.

Thoracic paravertebral sympathetic postganglionic neurons (tSPNs) comprise the final integrative output of the distributed sympathetic nervous system controlling vascular and thermoregulatory systems. Considered a non-integrating relay, what little is known of tSPN intrinsic excitability has been determined by sharp microelectrodes with presumed impalement injury. We thus undertook the first electrophysiological characterization of tSPN cellular properties using whole-cell recordings and coupled results with a conductance-based model to explore the principles governing their excitability in adult mice of both sexes. Recorded membrane resistance and time constant values were an order of magnitude greater than values previously obtained, leading to a demonstrable capacity for synaptic integration in driving recruitment. Variation in membrane resistivity was the primary determinant controlling cell excitability with vastly lower currents required for tSPN recruitment. Unlike previous microelectrode recordings in mouse which observed inability to sustain firing, all tSPNs were capable of repetitive firing. Computational modeling demonstrated that observed differences are explained by introduction of a microelectrode impalement injury conductance. Overall, tSPNs largely linearly encoded injected current magnitudes over a broad frequency range with distinct subpopulations differentiable based on repetitive firing signatures. Thus, whole-cell recordings reveal tSPNs have more dramatically amplified excitability than previously thought, with greater intrinsic capacity for synaptic integration and with the ability for maintained firing to support sustained actions on vasomotor tone and thermoregulatory function. Rather than acting as a relay, these studies support a more responsive role and possible intrinsic capacity for tSPNs to drive sympathetic autonomic function.

The role of the paravertebral ganglia in human sympathetic neural discharge patterns.

KEY POINTS: The mechanisms affecting recruitment patterns of postganglionic sympathetic nerves remain unclear. The divergent and convergent preganglionic innervation patterns of postganglionic neurons and the presence of differently sized postganglionic nerves suggest that the ganglia may participate in modifying the discharge patterns of single sympathetic postganglionic neurons innervating the skeletal muscle circulation. Whether the ganglia affect the ordered behaviour of varying sized postganglionic sympathetic neurons in humans has not been studied. Trimethaphan infusion produced an ordered pattern of action potential (AP) de-recruitment whereby the firing of larger, low probability APs present at baseline was abolished first, followed by progressive decreased probability of smaller APs. Although integrated sympathetic bursts were no longer detected after several minutes of trimethaphan, firing of the smallest APs was detected. These data suggest the ganglia affect the distribution of firing probabilities exhibited by differently sized sympathetic neurons. The ganglia may contribute to sympathetic neural emission patterns involved in homeostatic regulation. ABSTRACT: Do the ganglia contribute to the ordered behaviour of postganglionic neuronal discharge within the sympathetic nervous system? To further understand the functional organization of the sympathetic nervous system we employed the microneurographic approach to record muscle sympathetic nerve activity (MSNA) and a continuous wavelet transform to study postganglionic action potential (AP) behaviour during nicotinic blockade at the ganglia (trimethaphan camsylate, 1-7 mg min-1 ) in seven females (37 ± 5 years). Trimethaphan elicited a progressive reduction in sympathetic outflow characterized by fewer integrated bursts with decaying amplitude. Underlying trimethaphan-mediated attenuations in integrated MSNA were reductions in AP incidence (186 ± 101 to 29 ± 31 AP (100 beats)-1 ) and AP content per integrated burst (7 ± 2 to 3 ± 1 APs burst-1 ) (both P < 0.01) in the final minute of detectable bursting activity in the trimethaphan condition, compared to baseline. We observed an ordered de-recruitment of larger to smaller AP clusters active at baseline (14 ± 3 to 8 ± 2 active AP clusters, P < 0.01). Following cessation of integrated bursts in the trimethaphan condition, the smallest 6 ± 2 sympathetic AP clusters persisted to fire in an asynchronous pattern (49 ± 41 AP (100 beats)-1 ) in all participants. Valsalva's manoeuvre did not increase the incidence of these persistent APs (60 ± 42 AP (100 beats)-1 , P = 0.52), or recruit any larger APs in six of seven participants (6 ± 1 total AP clusters, P = 0.30). These data suggest that the ganglia participate in the ordered recruitment of differently sized postganglionic sympathetic nerves.

Physiological and pathophysiological firing properties of single postganglionic sympathetic neurons in humans.

It has long been known from microneurographic recordings in human subjects that the activity of postganglionic sympathetic axons occurs as spontaneous bursts, with muscle sympathetic nerve activity (MSNA) exhibiting strong cardiac rhythmicity via the baroreflex and skin sympathetic nerve activity showing much weaker cardiac modulation. Here we review the firing properties of single sympathetic neurons, obtained using highly selective microelectrodes. Individual vasoconstrictor neurons supplying muscle or skin, or sudomotor neurons supplying sweat glands, always discharge with a low firing probability (~30%) and at very low frequencies (~0.5 Hz). Moreover, they usually fire only once per cardiac interval but can fire greater than four times within a burst. Modeling has shown that this pattern can best be explained by individual neurons being driven by, on average, two preganglionic inputs. Unitary recordings of muscle vasoconstrictor neurons have been made in several pathophysiological states, including heart failure, hypertension, obstructive sleep apnea, bronchiectasis, chronic obstructive pulmonary disease, depression, and panic disorder. The augmented MSNA in each of these diseases features an increase in firing probability and discharge frequency of individual muscle vasoconstrictor neurons above that seen in healthy subjects, yet firing rates rarely exceed 1 Hz. However, unlike patients with heart failure, all patients with respiratory disease or panic disorder, and patients with hyperhidrosis, exhibited an increase in multiple within-burst firing, which emphasizes the different modes by which the sympathetic nervous system grades its output in pathophysiological states of high sympathetic nerve activity.

Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states.

KEY POINTS: Cardiac sympathetic afferents are considered to be essential pathways for transmission of cardiac nociception to the central nervous system during myocardial ischaemia. However, a potential contribution of the CSAR control of cardiac dysfunction in both normal and chronic heart failure (CHF) states remains unknown. We found that activation of the CSAR evokes little increase in cardiac contractility with an exaggerated peripheral vasoconstriction in the CHF state. CSAR inhibition by epicardial lidocaine decreased cardiac contractility to a greater extent in CHF rats than sham rats. Furthermore, we also found that epicardial lidocaine paradoxically decreased left ventricular end-diastolic pressure (LVEDP) and left ventricular end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. Chronic ablation of the CSAR by epicardial application of the afferent neurotoxin, RTX, selectively lowered diastolic blood pressure CHF rats. The observation suggests that CSAR has a differential effect on cardiac function in normal and CHF states. CSAR activation in normal state causes significant increase in cardiac contractility and cardiac output. ABSTRACT: The enhanced 'cardiac sympathetic afferent reflex' (CSAR) critically contributes to the exaggerated global sympathetic tone in chronic heart failure (CHF). However, a potential contribution of the cardio-cardiac reflex control of cardiac function in both normal and CHF states remains unknown. In this study, we evaluated the effects of direct activation or inhibition of the CSAR on cardiac function by pressure-volume (P-V) loop analysis in ∼12-week sham-operated and myocardial infarcted (MI) rats. In sham rats, acute CSAR activation by epicardial application of bradykinin (BK) increased heart rate (HR), left ventricular systolic pressure (LVSP), the maximum first derivative of left ventricular pressure (dp/dtmax ), and the slope of the end-systolic P-V relationship (ESPVR), suggesting that acute CSAR activation in the normal state enhances myocardial contractility. CSAR activation also decreased left ventricular (LV) systolic and diastolic volumes with little effect on LV end-diastolic pressure (LVEDP) or the end-diastolic P-V relationship (EDPVR) in sham rats. Compared to sham, CHF rats exhibit a reduced increase in the slope of the ESPVR and dp/dtmax in response to BK, indicating a poor contractile response to CSAR activation. Interestingly, BK application in CHF rats increased cardiac systolic and diastolic volumes and further increased the elevated LVEDP, neither of which was seen in sham rats. Following CSAR inhibition by epicardial lidocaine, blood pressure, HR, LVSP, dp/dt, LVEDP and ESPVR decreased in CHF rats whereas lidocaine had little effect in sham rats, indicating that the CSAR is tonically active in CHF and contributes to cardiac dysfunction. Furthermore, we found that epicardial lidocaine paradoxically decreased LV end-diastolic volume (preload) in CHF rats, which was not observed in sham rats. The decreased preload by lidocaine in CHF rats may be due to a reduction in peripheral vascular resistance since epicardial lidocaine significantly lowered peripheral (renal) sympathetic nerve activity in CHF rats but not in sham rats. Furthermore, chronic ablation of CSAR by epicardial application of a selective afferent neurotoxin, resiniferatoxin, selectively lowered diastolic blood pressure both at daytime and night-time with less effect on systolic blood pressure in CHF rats. Our data suggest that there is an imbalance between cardiac and peripheral responses to CSAR in CHF animals compared to sham-operated controls.

Carotid sinus denervation ameliorates renovascular hypertension in adult Wistar rats.

KEY POINTS: Peripheral chemoreflex sensitization is a feature of renovascular hypertension. Carotid sinus nerve denervation (CSD) has recently been shown to relieve hypertension and reduce sympathetic activity in other rat models of hypertension. We show that CSD in renovascular hypertension halts further increases in blood pressure. Possible mechanisms include improvements in baroreceptor reflex sensitivity and renal function, restoration of cardiac calcium signalling towards control levels, and reduced neural inflammation. Our data suggest that the peripheral chemoreflex may be a viable therapeutic target for renovascular hypertension. ABSTRACT: The peripheral chemoreflex is known to be hyper-responsive in both spontaneously hypertensive (SHR) and Goldblatt hypertensive (two kidney one clip; 2K1C) rats. We have previously shown that carotid sinus nerve denervation (CSD) reduces arterial blood pressure (ABP) in SHR. In the present study, we show that CSD ameliorates 2K1C hypertension and reveal the potential underlying mechanisms. Adult Wistar rats were instrumented to record ABP via telemetry, and then underwent CSD (n = 9) or sham CSD (n = 9) 5 weeks after renal artery clipping, in comparison with normal Wistar rats (n = 5). After 21 days, renal function was assessed, and tissue was collected to assess sympathetic postganglionic intracellular calcium transients ([Ca2+ ]i ) and immune cell infiltrates. Hypertensive 2K1C rats showed a profound elevation in ABP (Wistar: 98 ± 4 mmHg vs. 2K1C: 147 ± 8 mmHg; P < 0.001), coupled with impairments in renal function and baroreflex sensitivity, increased neuroinflammatory markers and enhanced [Ca2+ ]I in stellate neurons (P < 0.05). CSD reduced ABP in 2K1C+CSD rats and prevented the further progressive increase in ABP seen in 2K1C+sham CSD rats, with a between-group difference of 14 ± 2 mmHg by week 3 (P < 0.01), which was accompanied by improvements in both baroreflex control and spectral indicators of cardiac sympatho-vagal balance. Furthermore, CSD improved protein and albuminuria, decreased [Ca2+ ]i evoked responses from stellate neurons, and also reduced indicators of brainstem inflammation. In summary, CSD in 2K1C rats reduces the hypertensive burden and improves renal function. This may be mediated by improvements in autonomic balance, functional remodelling of post-ganglionic neurons and reduced inflammation. Our results suggest that the peripheral chemoreflex may be considered as a potential therapeutic target for controlling renovascular hypertension.