Superior Cervical Ganglion Research Articles

Selective Peripheral Chemoreceptor Hyperreflexia Evoked after Sympathetic Stimulation

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P<0.05; differences were tested using paired student‐t test.The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.Support or Funding InformationHealth Research Council of New Zealand

Effect of Superior Cervical Gangionectomy on the Chemoreflex Response to Hypoxia in the Rat Carotid Body

Carotid body (CB) chemosensitive glomus (type I) cells respond to decreases in blood pO2 (hypoxia) by releasing neurotransmitters, which in turn activate adjacent chemosensory afferent fibers within the carotid sinus nerve (CSN). These CSN chemoafferents then signal to the commissural nucleus tractus solitarius (cNTS) within the dorsal brainstem, which leads to a compensatory increase in ventilation in order to restore blood‐gas homeostasis. The CB receives sympathetic innervation via the ganglioglomerular nerve (GGN), whose cell bodies reside in the superior cervical ganglion (SCG). We have found that electrical stimulation of the GGN causes an increase in CB chemosensory afferent discharge. However, it is unknown the effect removing the carotid body’s sympathetic innervation, via superior cervical ganglionectomy, will have on the carotid body’s chemoreflex response to hypoxia. We hypothesize that loss of sympathetic input will decrease the hypoxic ventilatory response (HVR) of the rat, and alter the O2‐sensing mechanism of the CB chemosensitive glomus cells. Sprague Dawley male rats postnatal day 21 were subjected to unilateral or bilateral superior cervical ganglionectomy (SCGX). Four days post surgery (post‐natal day 25), rats either underwent whole body plethysmography recordings or were sacrificed and their carotid bodies removed to either isolate glomus cells and perform whole cell perforated patch clamp, or perform immunohistochemistry on the CB. Our preliminary results show that unilateral (left or right) SCGX appears to decrease the HVR during a 1‐minute exposure to hypoxia (10% O2, 90% N2) compared to bilateral SCGX. Our results also show that following SCGX, isolated glomus cells responded to acute hypoxia (HX) (5–8% O2, 5% CO2, balanced N2) with significant reductions (P<0.05 to P<0.01) in outward K+ current amplitudes over the test potential range +10 to +60 mV. The mean maximal effect, a reduction in K+ amplitude of 64.9 ± 18.5% (mean ± SD, n = 3 cells), occurred at a Vtest of +30mV. In contrast, control carotid body glomus cells responded to acute HX with a significant reduction (P<0.05) in outward K+ current amplitude at a Vtest of +60mV (n = 4 cells). In conclusion, our data suggests that sympathetic innervation to the CB is critical for the ventilatory response to hypoxia, and the O2‐sensing mechanism at the level of the CB glomus cells. Overall this study provides novel evidence for the importance of sympathetic innervation on peripheral chemosensors during exposure to hypoxia.Support or Funding InformationThis work was supported in part by a SPARC NIH award OT2OD023860

Effects of Central Intervention of Tumor Necrosis Factor‐α Converting Enzyme (TACE) on Neuroinflammation and Cardiac Function in Heart Failure

Central nervous system inflammation has been implicated in the neurohumoral activation and disease progression in experimental animal models of heart failure (HF). The pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) increases in cardiovascular/autonomic brain regions including the paraventricular nucleus of the hypothalamus (PVN) and subfornical organ (SFO), and contributes significantly to the augmented sympathetic nerve activity in HF. TNF‐α is initially synthesized as a membrane bound protein that is proteolytically cleaved by TNF‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease 17 (ADAM17), to become the fully functional inflammatory mediator. Our previous study demonstrated that TACE has elevated expression in the PVN and SFO and mediates TNF‐α‐induced sympathetic excitation and hemodynamic responses in HF rats. However, whether inhibition of TACE in the brain has a protective effect on central inflammation and cardiac function in HF remains unclear. We hypothesized that inhibition of TACE activity in the brain reduces central inflammation and improves left ventricular (LV) function during HF. Adult male Sprague‐Dawley rats were studied 4–5 weeks after induction of HF by coronary artery ligation. TACE activity was chronically inhibited specifically in the brain by infusion of the TACE inhibitor TAPI‐1 (30μg/day) via an osmotic minipump connected to an intracerebroventricular (ICV) cannula. Treatment with TAPI‐1 or vehicle began immediately following the myocardial infarction. At the end of experiments, LV function was assessed by echocardiography and tissues were collected for molecular analysis. In HF rats‐treated with TAPI‐1, the LV ejection fraction (26.8±0.9% vs 34.8±6.4%) was improved, although this did not reach statistical significance vs. HF treated with vehicle. Wet lung weight to body weight ratio also showed a trend towards being improved in TAPI‐1 treated animals (6.3±0.3mg/g vs. 4.8±0.4mg/g, vehicle vs. TAPI‐1, p=0.32). Additionally, TAPI‐1 treatment significantly decreased mRNA levels of proinflammatory mediators including TNF‐α, IL‐1β, and TACE in the SFO (TNF‐α: 0.28±0.10, p<0.05; IL1‐β: 0.25±0.11, p>0.05; TACE: 0.68±0.14, p<0.05, fold change vs. vehicle) and resulted in a trend toward reduced levels of these measurements in the PVN (TNF‐α: 0.52±0.13, p=0.24; IL1‐β: 0.58±0.11, p=0.21; TACE: 0.87±0.11, p=0.43, fold change vs. vehicle). TNF‐α expression was also reduced in both the superior cervical (0.35±0.07, p<0.05, fold change vs. vehicle) and stellate ganglia (0.44±0.14, p<0.05 fold change vs. vehicle). Taken together, these data suggest that central inhibition of TACE activity diminishes the inflammatory state in the brain and improve cardiac function in HF.

Neuropeptide Y may participate in the pathogenesis of cervical vertigo

ObjectivesThe concrete mechanism underlying the pathogenesis of cervical vertigo has not been well illustrated. This study aims to determine the diagnostic and potential therapeutic values of the neurotransmitter neuropeptide Y (NPY) in cervical vertigo. MethodsSixty male rabbits were randomly divided into five groups: the blank control, superior cervical sympathetic ganglia (SCSG) stimulation, SCSG sham-operated control (ganglion exposure and electrode placement in SCSG), inferior cervical sympathetic ganglia (ICSG) stimulation, and ICSG sham-operated control (ganglion exposure and electrode placement in ICSG) groups. Electrical stimulation at 30.0 Hz and 10.0 V for 5 min was delivered to expose SCSG or ICSG. Immunochemistry was conducted to determine the expression levels of NPY in all cervical spinal ganglia (C1–C8) dissected immediately after electrical stimulation. ResultsAccording to the immunohistochemical results, the blank control group only had few NPY-positive cells and was not significantly different with the sham-operated group. However, after SCSG stimulation, more NPY-positive cells in C2, C3, C4, and C5 were observed (C2 and C3 were the most evident). After ICSG stimulation, more NPY-positive cells were found in C6, C7, and C8, particularly in C6 and C7. ConclusionsThe low expression of NPY in all cervical ganglia (C1–C8) can be enhanced by electrical stimulation, whereas the expression of NPY in the cervical sympathetic ganglia was not affected by mechanical stimulation. This study provided new insights into the possible relationship between the neurotransmitter NPY and the pathogenesis of cervical vertigo.

Restoration of Cyclo-Gly-Pro-induced salivary hyposecretion and submandibular composition by naloxone in mice

Cyclo-Gly-Pro (CGP) attenuates nociception, however its effects on salivary glands remain unclear. In this study, we investigated the acute effects of CGP on salivary flow and composition, and on the submandibular gland composition, compared with morphine. Besides, we characterized the effects of naloxone (a non-selective opioid receptor antagonist) on CGP- and morphine-induced salivary and glandular alterations in mice. After that, in silico analyses were performed to predict the interaction between CGP and opioid receptors. Morphine and CGP significantly reduced salivary flow and total protein concentration of saliva and naloxone restored them to the physiological levels. Morphine and CGP also reduced several infrared vibrational modes (Amide I, 1687-1594cm-1; Amide II, 1594-1494cm-1; CH2/CH3, 1488-1433cm-1; C = O, 1432-1365cm-1; PO2 asymmetric, 1290-1185cm-1; PO2 symmetric, 1135-999cm-1) and naloxone reverted these alterations. The in silico docking analysis demonstrated the interaction of polar contacts between the CGP and opioid receptor Cys219 residue. Altogether, we showed that salivary hypofunction and glandular changes elicited by CGP may occur through opioid receptor suggesting that the blockage of opioid receptors in superior cervical and submandibular ganglions may be a possible strategy to restore salivary secretion while maintaining antinociceptive action due its effects on the central nervous system.

Reperfusion Arrhythmias Increase after Superior Cervical Ganglionectomy Due to Conduction Disorders and Changes in Repolarization.

Pharmacological concentrations of melatonin reduce reperfusion arrhythmias, but less is known about the antiarrhythmic protection of the physiological circadian rhythm of melatonin. Bilateral surgical removal of the superior cervical ganglia irreversibly suppresses melatonin rhythmicity. This study aimed to analyze the cardiac electrophysiological effects of the loss of melatonin circadian oscillation and the role played by myocardial melatonin membrane receptors, SERCA2A, TNFα, nitrotyrosine, TGFβ, KATP channels, and connexin 43. Three weeks after bilateral removal of the superior cervical ganglia or sham surgery, the hearts were isolated and submitted to ten minutes of regional ischemia followed by ten minutes of reperfusion. Arrhythmias, mainly ventricular tachycardia, increased during reperfusion in the ganglionectomy group. These hearts also suffered an epicardial electrical activation delay that increased during ischemia, action potential alternants, triggered activity, and dispersion of action potential duration. Hearts from ganglionectomized rats showed a reduction of the cardioprotective MT2 receptors, the MT1 receptors, and SERCA2A. Markers of nitroxidative stress (nitrotyrosine), inflammation (TNFα), and fibrosis (TGFβ and vimentin) did not change between groups. Connexin 43 lateralization and the pore-forming subunit (Kir6.1) of KATP channels increased in the experimental group. We conclude that the loss of the circadian rhythm of melatonin predisposes the heart to suffer cardiac arrhythmias, mainly ventricular tachycardia, due to conduction disorders and changes in repolarization.

Gait Deficits and Loss of Striatal Tyrosine Hydroxlase/Trk-B are Restored Following 7,8-Dihydroxyflavone Treatment in a Progressive MPTP Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is caused by neurodegeneration of nigrostriatal neurons, resulting in dopamine (DA) stimulated motor deficits. Like brain derived neurotrophic factor (BDNF), 7,8-dihydroxyflavone (DHF) is an agonist of the tropomyosin receptor kinase-B (TrkB) and stimulates the same secondary cascades that promote neuronal growth, survival and differentiation. We used our progressive mouse model of PD by administering increasing doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over 4 weeks (5 days/week), and then treated mice with DHF for 4 weeks after the cessation of the toxin injections (i.e., restoration). Mice treated with DHF recovered motorically, even after MPTP administration. Despite a 75% loss of tyrosine hydroxylase (TH) expression in the dorsolateral (DL) striatum in the MPTP group, mice treated with DHF had a recovery comparable to that found in the respective control. There was no recovery of DA tissue levels within the DL striatum. In both the DL striatum and substantia nigra (SN)/midbrain, phosphorylated TrkB and secondary messengers were significantly increased following DHF compared to the MPTP only group. Expression of the sprouting biomarker, superior cervical ganglion 10 (SCG10), was increased ∼20% in the DL striatum and 66% in the SN/midbrain in mice treated with DHF compared to the MPTP only group. We report that after 4 weeks of progressive MPTP administration, DHF can restore motor deficits and TH within the DL striatum in a TrkB-dependent manner. Our data suggests that DHF may help alleviate motor symptoms of PD and restore the loss of DA terminals within the striatum.

Inhibition of vascular adventitial remodeling by netrin-1 in diabetic rats.

Cardiovascular complications of type 2 diabetes mellitus (T2DM) are associated with vascular remodeling in the arteries. Perivascular sympathetic neurons release an abundance of trophic factors to regulate vascular function via a paracrine signaling. Netrin-1, a diffusible protein that can be secreted outside the cell, is one of common signals of 'conversation' between nerve and vessel. The present study investigated whether netrin-1 is a novel modulator of sympathetic neurons paracrine signaling and played a critical role in vascular adventitial remodeling under T2DM. Vascular adventitial remodeling was observed in adventitial fibroblasts (AFs) responding to netrin-1 deficiency in the supernatant from primary rat superior cervical ganglia (SCG) neurons, shown as AFs proliferation, migration, and collagen deposition. Conditioned medium from the high glucose (HG)-treated SCG neurons contributed to AFs remodeling, which was effectively alleviated by exogenous netrin-1 supplementation. Further, it was found that uncoordinated-5-B (Unc5b) was mainly expressed in AFs among netrin-1 specific receptors. Treatment of netrin-1 inhibited H2O2 production derived from NADPH oxidase 4 (NOX4) through the UNC5b/CAMP/PKA signal pathway in AFs remodeling. In vivo, aorta adventitial remodeling was accompanied with the downregulation of netrin-1 in the perivascular sympathetic nerve in T2DM rats. Such abnormalities were restored by netrin-1 intervention, which was associated with the inhibition of NOX4 expression in the aorta adventitia. In conclusion, netrin-1 is a novel modulator of sympathetic neurons paracrine signaling to maintain AFs function. Vascular adventitial remodeling was aggravated by sympathetic neurons paracrine signaling under hyperglycemia, which was ameliorated by netrin-1 treatment through the UNC5b/CAMP/PKA/NOX4 pathway.

Valeric Acid, a Gut Microbiota Product, Penetrates to the Eye and Lowers Intraocular Pressure in Rats.

Mechanisms controlling intraocular pressure (IOP) and arterial blood pressure (BP) share similar mediators, including gut bacteria metabolites. Here, we investigated the effects of valeric acid (VA), a short chain fatty acid produced by microbiota from undigested carbohydrates, on IOP and BP. To test if gut VA penetrates to the eye we evaluated eyes’ homogenates after the administration of D9-VA into the colon. Additionally, the following experimental series were performed on 16-week-old Sprague Dawley rats to analyze the influence of VA on IOP: vehicle treatment; VA treatment; VA + hydroxybutyrate - a short chain fatty acids’ G protein-coupled receptor 41/43 (GPR 41/43) blocker (ANT); hydroxybutyrate; VA + angiotensin II; angiotensin II; VA treatment in rats with superior cervical ganglion excision and sham operated rats. D9-VA rapidly penetrated from the colon to the eye. VA significantly decreased IOP and BP. The decrease in IOP was gradual and lasted through the experiment. In contrast, a decrease in BP was instantaneous and lasted no longer than 10 min. Angiotensin II, ANT, and sympathetic denervation did not influence the effect of VA on IOP. In conclusion, colon-derived VA penetrates to the eye and decreases IOP. The effect is independent from BP changes, angiotensin II, GPR41/43, and sympathetic eye innervation.

Magnetic resonance imaging features of the superior cervical ganglion and expected changes after radiation therapy to the head and neck in a long-term follow-up.

This study aimed to assess the magnetic resonance (MRI) features of the superior cervical ganglion (SCG) and to track changes to it induced using radiotherapy across a long-term follow-up. In total, 75 patients who underwent radiotherapy for head and neck malignancies and who were studied with MRI were recruited from two centers. MRI was performed before and after radiotherapy, with a median long-term follow-up of 4.5years. Baseline SCG features were assessed. Changes in axial cross-sectional area, T2-normalized signal, and apparent diffusion coefficient (ADC) (the latter available in about half of the patients) were analyzed. Repeated measures analysis of variance with Bonferroni's correction was used to analyze changes in the aforementioned parameters (significance level 0.05). Out of a potential 149 SCGs, 136 were visible at baseline MRI. A variable spatial relationship with the internal carotid artery was found. SCGs showed the "black dot" sign in almost all of the patients. ADC was higher in SCGs than in regional lymph nodes. Cross-sectional area, normalized T2, and ADC increased in the period up to 1year after radiotherapy and then remained stable in subsequent longer-term follow-up. The SCG has unusual features that allow differentiation from the regional lymph nodes. Changes in morphology and signal after radiotherapy must be taken into account by radiologists to avoid misdiagnosis as recurrent nodal disease. Changes induced using radiotherapy are stable in long-term follow-up and are thus likely attributed to other factors (such as Schwann cell hypertrophy/proliferation) rather than edema.

Long-term potentiation is differentially expressed in rostral and caudal neurons in the superior cervical ganglion of normal and hypertensive rats.

Neurons in the superior cervical ganglia (SCG) are classified as rostral and caudal according to their regional locations. Although diverse phenotypes have been reported for these two subpopulations, differences in neuroplasticity, like long-term potentiation (LTP), have not been characterized. Here, we explored possible regional differences of LTP expression in rostral and caudal neurons of the SCG in control rats, Wistar and Wistar Kyoto (WKy), and in the spontaneously hypertensive rats (SHR) as a model of hypertension. We characterized the expression of gLTP evoked by a tetanic train (40 Hz, 3 s) in an in vitro SCG preparation. gLTP was recorded in rostral and caudal neurons at 8-weeks-old (wo) in Wistar rats, 6-wo and 12-wo in SHR and WKy rats. We found that gLTP was differentially expressed; gLTP was larger in caudal neurons in Wistar and adult WKy rats. In adult 12-wo hypertensive SHR, gLTP was expressed in caudal but not in rostral neurons. In contrast, in 6-wo pre-hypertensive SHR, gLTP was expressed in rostral but not in caudal neurons; while in 6-wo WKy, gLTP was expressed in caudal but not in rostral neurons. The lack of gLTP expression in caudal neurons of 6-wo SHR was not due to a GABAergic modulation because several GABA-A receptor antagonists failed to unmask gLTP. Data show that neuroplasticity, particularly gLTP expression, varied according to the ganglionic region. We propose that differential regional expression of gLTP may be correlated with selective innervation on different target organs.

Carotid body type I cells engage flavoprotein and Pin1 for oxygen sensing.

Carotid body (CB) type I cells sense the blood Po2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN-, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND.

Sympathetic Enhancement of Memory T-Cell Homing and Hypertension Sensitization.

Effector memory T lymphocytes (TEM cells) exacerbate hypertension in response to repeated hypertensive stimuli. These cells reside in the bone marrow for prolonged periods and can be reactivated on reexposure to the hypertensive stimulus. Because hypertension is associated with increased sympathetic outflow to the bone marrow, we hypothesized that sympathetic nerves regulate accumulation and reactivation of bone marrow-residing hypertension-specific TEM cells. Using unilateral superior cervical ganglionectomy in wild-type C57BL/6 mice, we showed that sympathetic nerves create a bone marrow environment that supports residence of hypertension-specific CD8+ T cells. These cells, defined by their proliferative response on coculture with dendritic cells from Ang (angiotensin) II-infused mice, were reduced in denervated compared with innervated bone of Ang II-infused mice. Adoptively transferred CD8+ T cells from Ang II-infused mice preferentially homed to innervated compared with denervated bone. In contrast, ovalbumin responsive T cells from OT-I mice did not exhibit this preferential homing. Increasing superior cervical ganglion activity by activating Gq-coupled designer receptor exclusively activated by designer drug augmented CD8+ TEM bone marrow accumulation. Adoptive transfer studies using mice lacking β2AR (β2 adrenergic receptors) indicate that β2AR in the bone marrow niche, rather than T-cell β2AR is critical for TEM cell homing. Inhibition of global sympathetic outflow using Gi-coupled DREADD (designer receptor exclusively activated by designer drug) injected into the rostral ventrolateral medulla or treatment with a β2AR antagonist reduced hypertension-specific CD8+ TEM cells in the bone marrow and reduced the hypertensive response to a subsequent response to low dose Ang II. Sympathetic nerves contribute to the homing and survival of hypertension-specific TEM cells in the bone marrow after they are formed in hypertension. Inhibition of sympathetic nerve activity and β2AR blockade reduces these cells and prevents the blood pressure elevation and renal inflammation on reexposure to hypertension stimuli.

PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Maintenance of CaV2.2 channel-current by PIP2 unveiled by neomycin in sympathetic neurons of the rat

Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a native membrane phospholipid, has been involved in the maintenance of the current amplitude and in the voltage-independent regulation of voltage-gated calcium channels (VGCC). However, the nature of the PIP2 regulation on VGCC has not been fully elucidated. This work aimed to investigate whether the interacting PIP2 electrostatic charges may account for maintaining the current amplitude of CaV2.2 channels. Furthermore, we tested whether charge shielding of PIP2 mimics the voltage-independent inhibition induced by M1 muscarinic acetylcholine receptor (M1R) activation. Therefore, neomycin, a polycation that has been shown to block electrostatic interactions of PIP2, was intracellularly dialyzed in superior cervical ganglion (SCG) neurons of the rat. Consistently, neomycin time-dependently diminished the calcium current amplitude letting the channel exhibit the hallmarks of the voltage-independent regulation. These results support that interacting PIP2 charges not only underly the maintenance of the channel-current but also that charge screening of PIP2 by itself unveils the voltage-independent features of CaV2.2 channels in SCG neurons.

Axon Degeneration Assays in Superior Cervical Ganglion Explant Cultures.

The ability of peripheral nervous system neurons to extend long, axon-like neurites in vitro makes them ideally suited for studies on mechanisms of axon survival and degeneration. In this chapter, we describe how to prepare explant cultures of sympathetic neurons of the superior cervical ganglion (SCG). We also describe how to induce and assess axon degeneration with an injury or a chemical insult.

Microinjection of Superior Cervical Ganglion Neurons for Studying Axon Degeneration.

Primary cultures of neurons of the peripheral nervous system have been successfully used for studying many aspects of neuronal development and survival, including investigations into the mechanisms of axon degeneration. In this chapter, we describe how to prepare and microinject dissociated cultures of sympathetic neurons of the superior cervical ganglion (SCG) specifically for use in highly controlled and targeted assays of axon survival and degeneration.

Molecular and cellular mechanisms of the organogenesis and development of the mammalian carotid body.

Despite significant advancements in understanding physiological properties of the carotid body, little attention has been paid to its organogenesis. This review addresses the molecular and cellular mechanisms underlying organogenesis of the carotid body in mammals. The carotid body consists of two types of cells, that is, glomus cells and sustentacular cells, that are derived from different origins. Glomus cells are derivatives of neural crest cells which form sympathetic ganglia. Sustentacular cells are derivatives of mesenchymal neural crest cells which colonize the third pharyngeal arch and form the wall of the third arch artery. Gene-targeting studies indicate that three elements are required for carotid body organogenesis: the carotid sinus nerve (CSN), third arch artery, and superior cervical sympathetic ganglion (SCG). The CSN sends sensory fibers and Schwann cells to the wall of the third arch artery. The third arch artery provides mesenchymal cells, which give rise to sustentacular cells. The nerve process from the SCG sends glomus cell progenitors into the carotid body primordium. The presence of stem cells in the adult carotid body was recently highlighted. The origin of stem cells, however, remains controversial. Based on embryonic development of the carotid body, this review proposes the origin of stem cells.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BackgroundThe presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies – a question raised frequently due to their large size.Material and methodsRats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594.ResultsIn rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections.DiscussionOur inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Diabetes mellitus alters electrophysiological properties in neurons of superior cervical ganglion of rats

Diabetic neuropathy is the most prevalent complication associated with diabetes mellitus (DM). The superior cervical ganglion (SCG) is an important sympathetic component of the autonomic nervous system. We investigated the changes in cellular electrophysiological properties and on Na+K+-ATPase activity of SCG neurons of rats with DM induced by streptozotocin (STZ). Three types of action potentials (AP) firing pattern were observed in response to a long (1 s) depolarizing pulse. Whilst some neurons fired a single AP (single firing phasic, SFP), others fired few APs (multiple firing phasic, MFP). A third type fired APs during more than 80% of the stimulus duration (tonic-like, TL). The occurrence of SFP, MFP and TL was 84.5, 13.8, and 1.7%, respectively. SFP and MFP differed significantly in their membrane input resistance (Rin). At the end of the 4th week of its time course, DM differently affected most types of neurons: DM induced depolarization of resting membrane potential (RMP), decreased AP amplitude in SFP, and decreased Rin in MFP. DM decreased spike after-hyperpolarization amplitude in MFP and the duration in SFP. Based on the RMP depolarization, we investigated the Na+K+-ATPase action and observed that DM caused a significant decrease in Na+K+-ATPase activity of SCG. In conclusion, we have demonstrated that DM affects several parameters of SCG physiology in a manner likely to have pathophysiological relevance.