Superior Cervical Ganglion Research Articles

Expression of vitamin D receptors in the superior cervical ganglia of rats

We investigated the role of vitamin D in the sympathetic nervous system including the distribution of vitamin D receptors (VDR), 1α-hydroxylase and 24-hydroxylase (CYP24) in neuronal subpopulations and satellite glia in the superior cervical ganglia (SCGs) of rats using immunohistochemistry. VDR immunoreactivity was observed in the cytoplasm and nucleus of nearly all neurons in the SCG. Intensity of VDR fluorescence was significantly greater in the cytoplasm of neuropeptide Y (NPY) negative somata compared to NPY positive neurons. Immunoreactivity for 1α-hydroxylase also was observed in the cytoplasm of all neurons of the SCG, but the intensity of fluorescence was less in the nuclei. To the contrary, the immunoreactivity for CYP24 was stronger in the nuclei, although it was present at lower intensity also in the cytoplasm of neurons. VDR and 1α-hydroxylase immunofluorescence was observed in many non-neuron cells, except satellite glial cells, which exhibited weak CYP24 immunofluorescence. Expression of VDRs and key metabolizing enzymes indicated the importance of vitamin D in the autonomic nervous system and the ability of sympathetic neurons to activate and deactivate vitamin D for its autocrine and paracrine roles.

Molecular and cellular identification of the immune response in peripheral ganglia following nerve injury

BackgroundNeuroinflammation accompanies neural trauma and most neurological diseases. Axotomy in the peripheral nervous system (PNS) leads to dramatic changes in the injured neuron: the cell body expresses a distinct set of genes known as regeneration-associated genes, the distal axonal segment degenerates and its debris is cleared, and the axons in the proximal segment form growth cones and extend neurites. These processes are orchestrated in part by immune and other non-neuronal cells. Macrophages in ganglia play an integral role in supporting regeneration. Here, we explore further the molecular and cellular components of the injury-induced immune response within peripheral ganglia.MethodsAdult male wild-type (WT) and Ccr2−/− mice were subjected to a unilateral transection of the sciatic nerve and axotomy of the superior cervical ganglion (SCG). Antibody arrays were used to determine the expression of chemokines and cytokines in the dorsal root ganglion (DRG) and SCG. Flow cytometry and immunohistochemistry were utilized to identify the cellular composition of the injury-induced immune response within ganglia.ResultsChemokine expression in the ganglia differed 48 h after nerve injury with a large increase in macrophage inflammatory protein-1γ in the SCG but not in the DRG, while C-C class chemokine ligand 2 was highly expressed in both ganglia. Differences between WT and Ccr2−/− mice were also observed with increased C-C class chemokine ligand 6/C10 expression in the WT DRG compared to C-C class chemokine receptor 2 (CCR2)−/− DRG and increased CXCL5 expression in CCR2−/− SCG compared to WT. Diminished macrophage accumulation in the DRG and SCG of Ccr2−/− mice was found compared to WT ganglia 7 days after nerve injury. Interestingly, neutrophils were found in the SCG but not in the DRG. Cytokine expression, measured 7 days after injury, differed between ganglion type and genotype. Macrophage activation was assayed by colabeling ganglia with the anti-inflammatory marker CD206 and the macrophage marker CD68, and an almost complete colocalization of the two markers was found in both ganglia.ConclusionsThis study demonstrates both molecular and cellular differences in the nerve injury-induced immune response between DRG and SCG and between WT and Ccr2−/− mice.

Guinea Pig as a Model to Study the Carotid Body Mediated Chronic Intermittent Hypoxia Effects.

Clinical and experimental evidence indicates a positive correlation between chronic intermittent hypoxia (CIH), increased carotid body (CB) chemosensitivity, enhanced sympatho-respiratory coupling and arterial hypertension and cardiovascular disease. Several groups have reported that both the afferent and efferent arms of the CB chemo-reflex are enhanced in CIH animal models through the oscillatory CB activation by recurrent hypoxia/reoxygenation episodes. Accordingly, CB ablation or denervation results in the reduction of these effects. To date, no studies have determined the effects of CIH treatment in chemo-reflex sensitization in guinea pig, a rodent with a hypofunctional CB and lacking ventilatory responses to hypoxia. We hypothesized that the lack of CB hypoxia response in guinea pig would suppress chemo-reflex sensitization and thereby would attenuate or eliminate respiratory, sympathetic and cardiovascular effects of CIH treatment. The main purpose of this study was to assess if guinea pig CB undergoes overactivation by CIH and to correlate CIH effects on CB chemoreceptors with cardiovascular and respiratory responses to hypoxia. We measured CB secretory activity, ventilatory parameters, systemic arterial pressure and sympathetic activity, basal and in response to acute hypoxia in two groups of animals: control and 30 days CIH exposed male guinea pigs. Our results indicated that CIH guinea pig CB lacks activity elicited by acute hypoxia measured as catecholamine (CA) secretory response or intracellular calcium transients. Plethysmography data showed that only severe hypoxia (7% O2) and hypercapnia (5% CO2) induced a significant increased ventilatory response in CIH animals, together with higher oxygen consumption. Therefore, CIH exposure blunted hyperventilation to hypoxia and hypercapnia normalized to oxygen consumption. Increase in plasma CA and superior cervical ganglion CA content was found, implying a CIH induced sympathetic hyperactivity. CIH promoted cardiovascular adjustments by increasing heart rate and mean arterial blood pressure without cardiac ventricle hypertrophy. In conclusion, CIH does not sensitize CB chemoreceptor response to hypoxia but promotes cardiovascular adjustments probably not mediated by the CB. Guinea pigs could represent an interesting model to elucidate the mechanisms that underlie the long-term effects of CIH exposure to provide evidence for the role of the CB mediating pathological effects in sleep apnea diseases.

The mechanism of botulinum A on Raynaud syndrome.

BackgroundBotulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud’s phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear.Materials and methodsWe tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron.ResultsBased on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/A, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by Western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons.ConclusionOur study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE.

Nitration of MnSOD in the Carotid Body and Adrenal Gland Induced by Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (CIH), main feature of obstructive sleep apnea, produces nitro-oxidative stress, which contributes to potentiate carotid body (CB) chemosensory discharges and sympathetic-adrenal-axis activity, leading to hypertension. The MnSOD enzymatic activity, a key enzyme on oxidative stress control, is reduced by superoxide-induced nitration. However, the effects of CIH-induced nitration on MnSOD enzymatic activity in the CB and adrenal gland are not known. We studied the effects of CIH on MnSOD protein and immunoreactive (MnSOD-ir) levels in the CB, adrenal gland and superior cervical ganglion (SCG), and on 3-nitrotyrosine (3-NT-ir), CuZnSOD (CuZnSOD-ir), MnSOD nitration, and its enzymatic activity in the CB and adrenal gland from male Sprague-Dawley rats exposed to CIH for 7 days. CIH increased 3-NT-ir in CB and adrenal gland, whereas MnSOD-ir increased in the CB and in adrenal cortex, but not in the whole adrenal medulla or SCG. CIH nitrated MnSOD in the CB and adrenal medulla, but its activity decreased in the adrenal gland. CuZnSOD-ir remained unchanged in both tissues. All changes observed were prevented by ascorbic acid treatment. Present results show that CIH for 7 days produced MnSOD nitration, but failed to reduce its activity in the CB, because of the increased protein level.

A Calcium-Dependent Chloride Current Increases Repetitive Firing in Mouse Sympathetic Neurons.

Ca2+-activated ion channels shape membrane excitability in response to elevations in intracellular Ca2+. The most extensively studied Ca2+-sensitive ion channels are Ca2+-activated K+ channels, whereas the physiological importance of Ca2+-activated Cl- channels has been poorly studied. Here we show that a Ca2+-activated Cl- currents (CaCCs) modulate repetitive firing in mouse sympathetic ganglion cells. Electrophysiological recording of mouse sympathetic neurons in an in vitro preparation of the superior cervical ganglion (SCG) identifies neurons with two different firing patterns in response to long depolarizing current pulses (1 s). Neurons classified as phasic (Ph) made up 67% of the cell population whilst the remainders were tonic (T). When a high frequency train of spikes was induced by intracellular current injection, SCG sympathetic neurons reached an afterpotential mainly dependent on the ratio of activation of two Ca2+-dependent currents: the K+ [IK(Ca)] and CaCC. When the IK(Ca) was larger, an afterhyperpolarization was the predominant afterpotential but when the CaCC was larger, an afterdepolarization (ADP) was predominant. These afterpotentials can be observed after a single action potential (AP). Ph and T neurons had similar ADPs and hence, the CaCC does not seem to determine the firing pattern (Ph or T) of these neurons. However, inhibition of Ca2+-activated Cl- channels with anthracene-9′-carboxylic acid (9AC) selectively inhibits the ADP, reducing the firing frequency and the instantaneous frequency without affecting the characteristics of single- or first-spike firing of both Ph and T neurons. Furthermore, we found that the CaCC underlying the ADP was significantly larger in SCG neurons from males than from females. Furthermore, the CaCC ANO1/TMEM16A was more strongly expressed in male than in female SCGs. Blocking ADPs with 9AC did not modify synaptic transmission in either Ph or T neurons. We conclude that the CaCC responsible for ADPs increases repetitive firing in both Ph and T neurons, and it is more relevant in male mouse sympathetic ganglion neurons.

Retrograde influences of SCG axotomy on uninjured preganglionic neurons

There is evidence that neuronal injury can affect uninjured neurons in the same neural circuit. The overall goal of this study was to understand the effects of peripheral nerve injury on uninjured neurons located in the central nervous system (CNS). As a model, we examined whether axotomy (transection of postganglionic axons) of the superior cervical ganglion (SCG) affected the uninjured, preganglionic neurons that innervate the SCG. At 7 days post-injury a reduction in choline acetyltransferase (ChAT) and synaptophysin immunoreactivity in the SCG, both markers for preganglionic axons, was observed, and this reduction persisted at 8 and 12 weeks post-injury. No changes were observed in the number or size of the parent cell bodies in the intermediolateral cell column (IML) of the spinal cord, yet synaptic input to the IML neurons was decreased at both 8 and 12 weeks post-injury. In order to understand the mechanisms underlying these changes, protein levels of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) were examined and reductions were observed at 7 days post-injury in both the SCG and spinal cord. Taken together these results suggest that axotomy of the SCG led to reduced BDNF in the SCG and spinal cord, which in turn influenced ChAT and synaptophysin expression in the SCG and also contributed to the altered synaptic input to the IML neurons. More generally these findings provide evidence that the effects of peripheral injury can cascade into the CNS and affect uninjured neurons.

Superior Cervical Ganglionectomy Affects the Cardiorespiratory Network of the In Situ Arterially Perfused Brainstem Preparation

Recently we found that the superior cervical ganglia projects to the nucleus tractus solitarii. Here, we report the effect of superior cervical ganglionectomy (SCGx) on cardiorespiratory activity in the in situ, arterially perfused brainstem preparation. For these studies, we used male Sprague‐Dawley rat pups (P25). Five days after either bilateral removal of superior cervical ganglia (n=14) or sham surgery (n=14), we recorded efferent activities of the phrenic nerve, cervical vagus nerve, and thoracic sympathetic chain and monitored heart rate and perfusion pressure. Baseline cardio‐respiratory patterns were recorded for 10 min to assess differences in patterning between the SCGx and sham groups. Then, we evoked the peripheral chemoreceptor reflex by injecting (intra‐arterially) sodium cyanide (100 μL of a 0.03% solution) and the baroreceptor reflex by increasing perfusion pressure (increasing the speed of the roller pump to its maximum). Baseline respiratory frequency was similar in the SCGx and sham group. However, the SCGx group had a greater but delayed response to chemoreceptor stimulation. Similarly, the SCGx group had a greater response to baroreceptor stimulation. Baseline heart rates (HR) were similar in both groups. For the SCGx group, HR decreased less than sham group during chemoreceptor stimulation; but, decreased more than the sham group during baroreceptor stimulation. Finally, the SCGx group required a greater perfusion pressure (PP) than the sham group during baseline; and had smaller increase in PP during chemoreceptor stimulation. These data indicate that superior cervical ganglia modulate vasomotor tone at rest and buffer cardio‐respiratory responses to chemoreceptor and baroreceptor reflexes.Support or Funding InformationNIH‐SPARC 1OT2OD023860‐02SJLewisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Cervical Sympathetic Chain Stimulation on Upper Airway Pressure is Dependent on Direct Post‐Ganglionic Innervation from the Superior Cervical Ganglion

BACKGROUNDWe have previously demonstrated that cervical sympathetic chain (CSC) stimulation improves airway patency in Zucker Fat rats (model of concentric airway collapse). Anatomical studies reveal the presence of two major post‐ganglionic projections to the head and neck arising from the superior cervical ganglion (External Carotid Nerve (ECN) and Internal Carotid Nerve (ICN)), however, their functional contribution to improve upper airway patency is unclear.AIMWe sought to investigate the contribution of the CSC‐SCG‐ECN and CSC‐SCG‐ICN pathways on lowering upper airway pressure (UAP) and cardiorespiratory systems.METHODSUrethane‐anaesthetized healthy male Sprague‐Dawley (SD) rats and pathological Zucker Fat (ZF) rats were used to measure UAP, ECG, blood pressure and diaphragmatic EMG while the intact CSC was stimulated (bilaterally and unilaterally). CSC stimulation mediated effects on UAP and cardiorespiratory parameters were then assessed following systematic transection of ECN and ICN branches.RESULTSWe present two novel findings. Firstly, in all cases of CSC stimulation (SD control UAP (mmHg): −27% bilateral, −20% right, −6% left; ZF control UAP (mmHg): −34% bilateral, −19% right, −22% left), ECN denervation had no significant effect on UAP reductions or hemodynamics in both healthy and disease rats. Secondly, bilateral denervation of the ICN abolished CSC mediated UAP reductions, while leaving hemodynamic effects intact. UAP mediated effects via unilateral CSC stimulation were of ipsilateral origin, as contralateral ICN denervation prior to ipsilateral denervation had no effect. Little to no impact of laterality of CSC stimulation and ICN transection was seen in hemodynamics or diaphragmatic EMG.CONCLUSIONFor the first time, we have demonstrated that CSC stimulation reductions on upper airway pressure are dependent on ipsilateral post‐ganglionic innervation via the ICN. However, the lack of effect on hemodynamics remains to be defined.Support or Funding InformationNIH1OT2OD023860‐01, Galvani BioelectronicsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the Superior Cervical Ganglion in Response to Hypoxia in Juvenile Rats

The superior cervical ganglion (SCG) sends sympathetic input to the carotid body (CB) via the ganglioglomerular nerve (GGN) in order to modulate carotid sinus nerve (CSN) chemosensory afferent discharge via controlling vascular tone. This chemoafferent discharge is then relayed to the nucleus tractus solitarius (NTS) where the information is processed resulting in a compensatory increase in respiration. Hypoxic gas challenge stimulates the GGN. However, little is known as to the effects of superior cervical ganglionecomy (SCGx) on carotid body chemosensory afferent response to hypoxia (HX).Our objective was to determine the effect of unilateral SCGx on the ventilatory responses elicited by HX (10% O2, 90% N2) gas challenge (5 episodes of 5 min in duration separated by 15 min) in freely‐moving juvenile (P25) Sprague‐Dawley rats.Male rats postnatal (P) 21 were subjected to sham or unilateral (right) SCGx and were allowed 4 days to recover. On the day of the experiment, P25 rats were placed in whole‐body plethysmography chambers and subjected to the hypoxic gas exposure. Ventilatory parameters included frequency of breathing, tidal volume, minute ventilation (= frequency x tidal volume), inspiratory and expiatory times, end inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50 (expiratory flow at 50% expired tidal volume) and central inspiratory drive (tidal volume/inspiratory time).The HX challenge elicited robust ventilatory responses in sham‐operated rats compared to unilateral (right) SCGx. Frequency (breathes/min), minute ventilation (ml/min), peak inspiratory flow (msec), and peak expiratory flow (msec) were diminished in unilateral (right) SCGx rats compared to sham. End expiratory pause (msec) showed a faster return to baseline following hypoxic challenge in unilateral (right) SCGx compared to sham. These findings demonstrate a role for CB sympathetic input from the SCG in modulating the ventilatory response to hypoxia.Support or Funding InformationNIH‐SPARC 1OT2OD023860‐02 SJLewisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Superior Cervical Ganglionectomy alters Glomus Cell Potassium‐Channel Properties in Response to Hypoxia

The carotid body (CB) chemosensory glomus (type I) cells respond to changes in blood PO2, PCO2, and pH by releasing neurotransmitters that increase action potential discharge of chemosensory afferent fibers within the carotid sinus nerve (CSN). The CSN then signals to the nucleus tractus solitarius (NTS) within the dorsal brainstem, which leads to a compensatory increase in ventilation in order to restore blood gas levels and pH. Along with the CSN, the ganglioglomerular nerve (GGN) also innervates the carotid body. The GGN consists of post‐ganglionic sympathetic fibers, which originate from the superior cervical ganglion (SCG). Stimulation of the GGN causes an increase in CB chemosensory afferent discharge. However, it is unknown whether loss of sympathetic input to the CB directly alters the chemosensitivity of glomus cells. Our objective was to characterize electrophysiological properties of CB glomus cell potassium (K+) channels in response to acute hypoxia following superior cervical ganglionectomy (SCGx). Sprague Dawley (SD) male rats postnatal (P) 21 were subjected to unilateral (right) SCGx. The left SCG was identified, but not removed. Four days post surgery at P25 the right and left carotid bodies were removed, and primary glomus cells were isolated and plated on coverslips. Glomus cell K+ channel properties were then recorded using the whole‐cell perforated patch‐clamp technique. Our preliminary results reveal that following unilateral (right) SCGx, glomus cells respond to hypoxia 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 +30 mV. In contrast, wild‐type control CB glomus cells respond to hypoxia with a significant reduction (P<0.05) in outward K+ current amplitude at only a Vtest of +60 mV (n = 4 cells). In addition, glomus cells from SCGx rats appear to respond to hypoxia via a different population of K+ channels that do not appear to be presently active in wild‐type glomus cells when exposed to hypoxia. These findings demonstrate that loss of sympathetic input from the SCG increases CB glomus cell sensitivity to hypoxia, and modulates the O2‐sensing capabilities of glomus cell K+ channels.Support or Funding InformationNIH‐SPARC 1OT2OD023860‐02 SJLewisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

When PNS Assembly Goes Awry: Familial Dysautonomia

Over a century of elegant work has contributed to our understanding of how neural crest cells migrate throughout the embryo to give rise to the majority of cell types in the peripheral nervous system (PNS). This rich foundation is the basis for our lab's investigation of the cellular and molecular mechanisms that go awry to result in the developmental disorder of the PNS, Familial dysautonomia (FD). Three of the key clinical hallmarks of FD include a reduction in pain and temperature sensation, misregulation of the sympathetic nervous system, and impaired gastrointestinal tract function. The neurons in all of these systems derive from the neural crest. Consequently, a prevailing hypothesis in the field was that FD resulted from faulty neural crest migration. To test this hypothesis, we generated mice in which the gene that is mutated to cause FD, IKBKAP/ELP1, was deleted selectively from the neural crest. Interestingly, by the end of embryogenesis, we found that in fact the number of pain and temperature receptors in the dorsal root ganglia were significantly reduced as were sympathetic neurons in the superior cervical ganglia, recapitulating the human disease phenotype (George et al., 2013, PNAS). However, these studies also demonstrated that this selective reduction in neuronal number was not due to faulty neural crest cell migration. Rather, the reductions were due to (1) the death and premature differentiation of the Pax3+ pain and temperature progenitor cells and (2) exacerbated apoptosis of TrkA+ pain and temperature receptors. Interestingly, the TrkC+ proprioceptors developed normally, although they later die progressively in adulthood (Elisabetta Morini and Susan Slaughenhaupt, personal communication). Upon the completion of neurogenesis in the dorsal root and sympathetic ganglia, PNS axons must innervate their targets to fully differentiate. Our work and others (Jackson et al., 2014, Development; Abashidze et al., 2015, PLoS One) demonstrate that IkbkapElp1 is required for normal axon outgrowth and target innervation. These and other findings for how and why PNS development is disrupted in FD will be discussed.Support or Funding InformationNIH R01 NS086796; Dysautonomia FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of mouse superior cervical ganglion during prenatal and postnatal stages.

The neuropeptide calcitonin gene‐related peptide (CGRP) is a well‐characterized mediator of receptor‐regulated neurotransmitter release. However, little is known between CGRP during the development of the superior cervical ganglion (SCG). In the present study, we observed the localization of the CGRP mRNA using in situ hybridization during development from the embryonic stage to after birth day 14.5 (E14.5), E17.5 and postnatal day 1 (P1). The different ratio was found in SCG ganglion cells and shrunken cells. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels of positive CGRP ganglion cells were detected at E17.5. The specific characters of CGRP concentration and release were occurred during development. The CGRP may influence the functional stress during prenatal and postnatal development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insights into the neurochemical signature of the Innervation of Beige Fat

ObjectiveThe potential for brown adipose tissue (BAT) to be targeted as a therapeutic option to combat obesity has been heightened by the discovery of a brown–like form of inducible “beige” adipose tissue in white fat which has overlapping structural and functional properties to “classical” BAT. The likelihood that both beige and brown fat are recruited functionally by neural mechanisms, taken together with the lack of a detailed understanding of the nature of changes in the nervous system when white adipose tissue (WAT) is transformed to brown, provides the impetus for this study. Here, we aim to identify whether there is a shift in the gene expression profile in neurons directly innervating inguinal white adipose tissue (iWAT) that has undergone “beiging” to a signature that is more similar to neurons projecting to BAT. MethodsTwo groups of rats, one housed at thermoneutrality (27 °C) and the other exposed to cold (8 °C) for 7 days, were killed, and their T13/L1 ganglia, stellate ganglion (T1/T2), or superior cervical ganglion (SCG, C2/3) removed. This approach yielded ganglia containing neurons that innervate either beiged white fat (8 °C for 7 days), inguinal WAT (27 °C for 7 days), BAT (both 27 °C and 8 °C for 7 days) or non-WAT (8 °C for 7 days), the latter included to isolate changes in gene expression that were more aligned with a response to cold exposure than the transformation of white to beige adipocytes. Bioinformatics analyses of RNA sequencing data was performed followed by Ingenuity Pathway Analysis (IPA) to determine differential gene expression and recruitment of biosynthetic pathways. ResultsWhen iWAT is “beiged” there is a significant shift in the gene expression profile of neurons in sympathetic ganglia (T13/L1) innervating this depot toward a gene neurochemical signature that is similar to the stellate ganglion projecting to BAT. Bioinformatics analyses of “beiging” related genes revealed upregulation of genes encoding neuropeptides proopiomelanocortin (POMC) and calcitonin-gene related peptide (CGRP) within ganglionic neurons. Treatment of differentiated 3T3L1 adipocytes with αMSH, one of the products cleaved from POMC, results in an elevation in lipolysis and the beiging of these cells as indicated by changes in gene expression markers of browning (Ucp1 and Ppargc1a). ConclusionThese data indicate that, coincident with beiging, there is a shift toward a “brown-like” neurochemical signature of postganglionic neurons projecting to inguinal white fat, an increased expression of POMC, and, consistent with a causative role for this prohormone in beiging, an αMSH-mediated increase in beige gene markers in isolated adipocytes.

The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

Superior cervical gangliectomy induces non-exudative age-related macular degeneration in mice.

ABSTRACTNon-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

Cholinergic responses of satellite glial cells in the superior cervical ganglia

Satellite glial cells (SGCs) surround the neurons in sympathetic ganglia and are believed to make important contributions to the function of the ganglia under normal and pathological conditions. It has been proposed that SGCs communicate chemically with the neurons, but little is known about their pharmacological properties and there is no information on whether they respond to acetylcholine (ACh), which is the major neurotransmitter in these ganglia. We used calcium imaging to examine responses of SGCs in the mouse superior cervical ganglion to ACh. The SGCs responded to ACh (0.01–2 mM) with an elevation of intracellular Ca2+, which appeared to be due to direct action on these cells, as the response persisted in the presence of the nerve blocker tetrodotoxin (1 μM). The response was largely inhibited by atropine, indicating an action on muscarinic ACh receptors. In contrast to this, sensory ganglia (nodose and trigeminal) were not sensitive to ACh. Incubation of the ganglia in ACh (0.5 or 1 mM) increased the expression of glial fibrillay acidic protein, which is a marker for glial activation. Such incubation also increased the electrical coupling of SGCs, which is known to occur in sensory ganglia following injury. We conclude that SGCs in the superior cervical ganglia display muscarinic ACh receptors, which enable them to communicate chemically with the sympathetic neurons.

Immunohistochemical localization of angiotensin AT1 receptors in the rat carotid body

The carotid body (CB) is a major peripheral arterial chemoreceptor that initiates respiratory and cardiovascular adjustments to maintain homeostasis. Recent evidence suggests that circulating or locally produced hormones like angiotensin II acting via AT1 receptors modulate its activity in a paracrine-autocrine manner. The aim of this study was to examine the immunohistochemical localization of AT1 receptor in the CB of adult rats and to compare its expression in vehicle-treated animals, and after the long-term application of its selective blocker losartan. Immunohistochemistry revealed that a subset of CB glomeruli and the vast majority of neurons in the adjacent superior cervical ganglion (SCG) were strongly AT1 receptor-immunoreactive. In the CB immunostaining was observed in the chemosensory glomus cells typically aggregated in cell clusters while the nerve fibers in-between and large capillaries around them were immunonegative. Exogenous administration of losartan for a prolonged time significantly reduces the intensity of AT1 receptor immunostaining in the CB glomus cells and SCG neurons. Our results show that AT1 receptors are largely expressed in the rat CB under physiological conditions, and their expression is down-regulated by losartan treatment.

Alterations in Metabolism and Diurnal Rhythms following Bilateral Surgical Removal of the Superior Cervical Ganglia in Rats.

Mammalian circadian rhythms are controlled by a master pacemaker located in the suprachiasmatic nuclei (SCN), which is synchronized to the environment by photic and nonphotic stimuli. One of the main functions of the SCN is to regulate peripheral oscillators to set temporal variations in the homeostatic control of physiology and metabolism. In this sense, the SCN coordinate the activity/rest and feeding/fasting rhythms setting the timing of food intake, energy expenditure, thermogenesis, and active and basal metabolism. One of the major time cues to the periphery is the nocturnal melatonin, which is synthesized and secreted by the pineal gland. Under SCN control, arylalkylamine N-acetyltransferase (AA-NAT)—the main enzyme regulating melatonin synthesis in vertebrates—is activated at night by sympathetic innervation that includes the superior cervical ganglia (SCG). Bilateral surgical removal of the superior cervical ganglia (SCGx) is considered a reliable procedure to completely prevent the nocturnal AA-NAT activation, irreversibly suppressing melatonin rhythmicity. In the present work, we studied the effects of SCGx on rat metabolic parameters and diurnal rhythms of feeding and locomotor activity. We found a significant difference between SCGx and sham-operated rats in metabolic variables such as an increased body weight/food intake ratio, increased adipose tissue, and decreased glycemia with a normal glucose tolerance. An analysis of locomotor activity and feeding rhythms showed an increased daytime (lights on) activity (including food consumption) in the SCGx group. These alterations suggest that superior cervical ganglia-related feedback mechanisms play a role in SCN-periphery phase coordination and that SCGx is a valid model without brain-invasive surgery to explore how sympathetic innervation affects daily (24 h) patterns of activity, food consumption and, ultimately, its role in metabolism homeostasis.

Decreased intracellular granule movement and glucagon secretion in pancreatic α cells attached to superior cervical ganglion neurites

Autonomic neurons innervate pancreatic islets of Langerhans and participate in the maintenance of blood glucose concentrations by controlling hormone levels through attachment with islet cells. We previously found that stimulated superior cervical ganglia (SCG) could induce Ca2+ oscillation in α cells via neuropeptide substance P using an in vitro co-culture model. In this study, we studied the effect of SCG neurite adhesion on intracellular secretory granule movement and glucagon secretion in α cells stimulated by low glucose concentration. Spinning disk microscopic analysis revealed that the mean velocity of intracellular granules was significantly lower in α cells attached to SCG neurites than that in those without neurites under low (2mM), middle (10mM), and high (20mM) glucose concentrations. Stimulation by a low (2mM) glucose concentration significantly increased glucagon secretion in α cells lacking neurites but not in those bound to neurites. These results suggest that adhesion to SCG neurites decreases low glucose-induced glucagon secretion in pancreatic α cells by attenuating intracellular granule movement activity.