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O-GlcNAcylation is a post-translational modification (PTM) that regulates a wide range of cellular functions and has been associated with multiple metabolic diseases in various organs. The sympathetic nervous system (SNS) is the efferent portion of the autonomic nervous system that regulates metabolism of almost all organs in the body. How much the development and functionality of the SNS are influenced by O-GlcNAcylation, as well as how such regulation could contribute to sympathetic neuron (symN)-related neuropathy in diseased states, remains unknown. Here, we assessed the level of protein O-GlcNAcylation at various stages of symN development, using a human pluripotent stem cell (hPSC)-based symN differentiation paradigm. We found that pharmacological disruption of O-GlcNAcylation impaired both the growth and survival of hPSCderived symNs. In the high glucose condition that mimics hyperglycemia, hPSCderived symNs were hyperactive, and their regenerative capacity was impaired, which resembled typical neuronal defects in patients and animal models of diabetes mellitus. Using this model of sympathetic neuropathy, we discovered that O-GlcNAcylation increased in symNs under high glucose, which lead to hyperactivity. Pharmacological inhibition of O-GlcNAcylation rescued high glucose-induced symN hyperactivity and cell stress. This framework provides the first insight into the roles of O-GlcNAcylation in both healthy and diseased human symNs and may be used as a platform for therapeutic studies. [ABSTRACT FROM AUTHOR]

Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and this also holds true for e.g., P2Y12 antagonists. A new target at the horizon of cardiovascular drugs is the P2Y6 receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y6 receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y6 receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y6 receptor antagonist MRS2578, but not by the P2Y2 antagonist ARC118925XX. UDP raised intracellular Ca2+ in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl− conductances, and these were reduced by a blocker of Ca2+-activated Cl− channels, CaCCInh-A01. In addition, UDP inhibited currents through KV7 channels. The increase in numbers of action potentials caused by UDP was not altered by the KV7 channel blocker linopirdine, but was enhanced in low extracellular Cl− and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated [3H] noradrenaline, and this was augmented in low extracellular Cl− and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y6 receptor activation involving Ca2+- activated Cl− channels. [ABSTRACT FROM AUTHOR]

Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and this also holds true for e.g., P2Y12 antagonists. A new target at the horizon of cardiovascular drugs is the P2Y6 receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y6 receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y6 receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y6 receptor antagonist MRS2578, but not by the P2Y2 antagonist AR-C118925XX. UDP raised intracellular Ca2+ in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl− conductances, and these were reduced by a blocker of Ca2+-activated Cl− channels, CaCCInh-A01. In addition, UDP inhibited currents through KV7 channels. The increase in numbers of action potentials caused by UDP was not altered by the KV7 channel blocker linopirdine, but was enhanced in low extracellular Cl− and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated [3H] noradrenaline, and this was augmented in low extracellular Cl− and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y6 receptor activation involving Ca2+-activated Cl− channels.

The Spontaneously Hypertensive Rat (SHR) has increased sympathetic drive to the periphery that precedes and contributes to the development of high blood pressure, making it a useful model for the study of neurogenic hypertension. Comparisons to the normotensive Wistar Kyoto (WKY) rat have demonstrated altered active and intrinsic properties of SHR sympathetic neurons shortly before the onset of hypertension. Here we examine the structural and functional plasticity of postnatal SHR and WKY sympathetic neurons cultured alone or co-cultured with cardiomyocytes under conditions of limited extrinsic signaling. SHR neurons have an increased number of structural synaptic sites compared to age-matched WKY neurons, measured by the co-localization of presynaptic vesicular acetylcholine transporter and postsynaptic shank proteins. Whole cell recordings show that SHR neurons have a higher synaptic charge than WKY neurons, demonstrating that the increase in synaptic sites is associated with increased synaptic transmission. Differences in synaptic properties are not associated with altered firing rates between postnatal WKY and SHR neurons and are not influenced by interactions with target cardiomyocytes from either strain. Both SHR and WKY neurons show tonic firing patterns in our cultures, which are depleted of non-neuronal ganglionic cells and provide limited neurotrophic signaling. This suggests that the normal mature, phasic firing of sympathetic neurons requires extrinsic signaling, with potentially differential responses in the prehypertensive SHR, which have been reported to maintain tonic firing at later developmental stages. While cardiomyocytes do not drive neuronal differences in our cultures, SHR cardiomyocytes display decreased hypertrophy compared to WKY cells and altered responses to co-cultured sympathetic neurons. These experiments suggest that altered signaling in SHR neurons and cardiomyocytes contributes to changes in the cardiac-sympathetic circuit in prehypertensive rats as early as the postnatal period.

Satellite glia are the major glial cells in sympathetic ganglia, enveloping neuronal cell bodies. Despite this intimate association, the extent to which sympathetic functions are influenced by satellite glia in vivo remains unclear. Here, we show that satellite glia are critical for metabolism, survival, and activity of sympathetic neurons and modulate autonomic behaviors in mice. Adult ablation of satellite glia results in impaired mTOR signaling, soma atrophy, reduced noradrenergic enzymes, and loss of sympathetic neurons. However, persisting neurons have elevated activity, and satellite glia-ablated mice show increased pupil dilation and heart rate, indicative of enhanced sympathetic tone. Satellite glia-specific deletion of Kir4.1, an inward-rectifying potassium channel, largely recapitulates the cellular defects observed in glia-ablated mice, suggesting that satellite glia act in part via K+-dependent mechanisms. These findings highlight neuron-satellite glia as functional units in regulating sympathetic output, with implications for disorders linked to sympathetic hyper-activity such as cardiovascular disease and hypertension. [ABSTRACT FROM AUTHOR]

Satellite glia are the major glial cells in sympathetic ganglia, enveloping neuronal cell bodies. Despite this intimate association, the extent to which sympathetic functions are influenced by satellite glia in vivo remains unclear. Here, we show that satellite glia are critical for metabolism, survival, and activity of sympathetic neurons and modulate autonomic behaviors in mice. Adult ablation of satellite glia results in impaired mTOR signaling, soma atrophy, reduced noradrenergic enzymes, and loss of sympathetic neurons. However, persisting neurons have elevated activity, and satellite glia-ablated mice show increased pupil dilation and heart rate, indicative of enhanced sympathetic tone. Satellite glia-specific deletion of Kir4.1, an inward-rectifying potassium channel, largely recapitulates the cellular defects observed in glia-ablated mice, suggesting that satellite glia act in part via K+-dependent mechanisms. These findings highlight neuron–satellite glia as functional units in regulating sympathetic output, with implications for disorders linked to sympathetic hyper-activity such as cardiovascular disease and hypertension.

Propionic acid is a cell nutrient but also a stimulus for cellular signaling. Free fatty acid receptor (FFAR)-3, also known as GPR41, is a Gi/o protein-coupled receptor (GPCR) that mediates some of the propionate's actions in cells, such as inflammation, fibrosis, and increased firing/norepinephrine release from peripheral sympathetic neurons. The regulator of G-protein Signaling (RGS)-4 inactivates (terminates) both Gi/o- and Gq-protein signaling and, in the heart, protects against atrial fibrillation via calcium signaling attenuation. RGS4 activity is stimulated by β-adrenergic receptors (ARs) via protein kinase A (PKA)-dependent phosphorylation. Herein, we examined whether RGS4 modulates cardiac FFAR3 signaling/function. We report that RGS4 is essential for dampening of FFAR3 signaling in H9c2 cardiomyocytes, since siRNA-mediated RGS4 depletion significantly enhanced propionate-dependent cAMP lowering, Gi/o activation, p38 MAPK activation, pro-inflammatory interleukin (IL)-1β and IL-6 production, and pro-fibrotic transforming growth factor (TGF)-β synthesis. Additionally, catecholamine pretreatment blocked propionic acid/FFAR3 signaling via PKA-dependent activation of RGS4 in H9c2 cardiomyocytes. Finally, RGS4 opposes FFAR3-dependent norepinephrine release from sympathetic-like neurons (differentiated Neuro-2a cells) co-cultured with H9c2 cardiomyocytes, thereby preserving the functional βAR number of the cardiomyocytes. In conclusion, RGS4 appears essential for propionate/FFAR3 signaling attenuation in both cardiomyocytes and sympathetic neurons, leading to cardioprotection against inflammation/adverse remodeling and to sympatholysis, respectively. [ABSTRACT FROM AUTHOR]

Dendritic morphology is a critical determinant of neuronal connectivity, and in postganglionic sympathetic neurons, tonic activity correlates directly with the size of the dendritic arbor. Thus, identifying signaling mechanisms that regulate dendritic arborization of sympathetic neurons is important to understanding how functional neural circuitry is established and maintained in the sympathetic nervous system. Bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, downstream signaling events that link BMP receptor activation to dendritic growth are poorly characterized. We previously reported that BMP7 upregulates p75(NTR) mRNA in cultured sympathetic neurons. This receptor is implicated in controlling dendritic growth in central neurons but whether p75(NTR) regulates dendritic growth in peripheral neurons is not known. Here, we demonstrate that BMP7 increases p75(NTR) protein in cultured sympathetic neurons, and this effect is blocked by pharmacologic inhibition of signaling via BMP type I receptor. BMP7 does not trigger dendritic growth in sympathetic neurons dissociated from superior cervical ganglia (SCG) of p75(NTR) nullizygous mice, and overexpression of p75(NTR) in p75(NTR) -/- neurons is sufficient to cause dendritic growth even in the absence of BMP7. Morphometric analyses of SCG from wild-type versus p75(NTR) nullizygous mice at 3, 6, and 12 to 16 weeks of age indicated that genetic deletion of p75(NTR) does not prevent dendritic growth but does stunt dendritic maturation in sympathetic neurons. These data support the hypotheses that p75(NTR) is involved in downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that p75(NTR) signaling positively modulates dendritic complexity in sympathetic neurons in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1003-1013, 2016.

Sympathetic neurons deprived of nerve growth factor (NGF) die by apoptosis. Chronic depolarization with elevated concentrations of extracellular potassium ([K+]E) supports long-term survival of these and other types of neurons in culture. While depolarization has long been used to support neuronal cultures, little is known about the mechanism. We explored how chronic depolarization of NGF-deprived mouse sympathetic neurons in culture blocks apoptotic death. First, we determined the effects of elevated [K+]E on proapoptotic BH3-only proteins reported to be upregulated in sympathetic neurons after NGF withdrawal. Upregulation of BIMEL was blocked by depolarization while upregulation of PUMA was not. BMF levels did not increase after NGF withdrawal, and elevated [K+]E had no effect on its expression. dp5/HRK was not detectable. A large increase in production of mitochondria-derived reactive species (RS), including reactive oxygen species (ROS), occurs in NGF-deprived sympathetic neurons. Suppressing these RS prevents cytochrome c release from mitochondria and apoptosis. The addition of high [K+]E to cultures rapidly blocked increased RS and cytochrome c release. Elevated [K+]E caused an increase of the cellular antioxidant glutathione (GSH). Preventing this increase prevented the elevated [K+]E from blocking cytochrome c release and death. While suppression of BIMEL upregulation by elevated [K+]E may contribute to high [K+]E pro-survival effects, we conclude that elevated [K+]E prevents apoptotic death of NGF-deprived sympathetic neurons primarily via an antioxidant mechanism. [ABSTRACT FROM AUTHOR]

Yanwen Zhou,* Ying Liu,* Yunhua Hao, Ya Feng, Lizhen Pan, Wuchao Liu, Bing Li, Libin Xiao, Lingjing Jin, Zhiyu Nie Department of Neurology, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China *These authors contributed equally to this work Background: Botulinum 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 methods: We 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. Results: Based on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/Α, 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. Conclusion: Our 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. Keywords: botulinum neurotoxin type A, Raynaud’s phenomenon, α-adrenoceptor, arteriole diameter constrict rate, SNAP-25, SV2C, GM1, FGFR3, sympathetic neuron, vesicle cycle

The sympathetic nervous system plays critical roles in the differentiation, maturation and recruitment of immune cells under homeostatic conditions, and in responses to environmental stimuli, although its role in the migratory control of immune cells during acute inflammation remains unclear. In this study, using an advanced intravital bone imaging system established in our laboratory, we demonstrated that the sympathetic nervous system locally regulates neutrophil egress from the bone marrow for mobilization to inflammatory foci. We found that sympathetic neurons were located close to blood vessels in the bone marrow cavity; moreover, upon lipopolysaccharide (LPS) administration, local sympathectomy delayed neutrophil egress from the bone marrow and increased the proportion of neutrophils that remained in place. We also showed that vascular endothelial cells produced C-X-C motif chemokine ligand 1 (CXCL1), which is responsible for neutrophil egress out of the bone marrow. Its expression was up-regulated during acute inflammation, and was suppressed by β-adrenergic receptor blockade, which was accompanied with inhibition of neutrophil egress into the systemic circulation. Furthermore, systemic β-adrenergic signaling blockade decreased the recruitment of neutrophils in the lung under conditions of acute systemic inflammation. Taken together, the results of this study first suggested a new regulatory system, wherein local sympathetic nervous activation promoted neutrophil egress by enhancing Cxcl1 expression in bone marrow endothelial cells in a β-adrenergic signaling-dependent manner, contributing to the recruitment of neutrophils at the onset of inflammation in vivo. [ABSTRACT FROM AUTHOR]

This study was designed to determine whether and how the sympathetic nervous system (SNS) regulates motoneuron axon function and neuromuscular transmission in young (3-4-month) and geriatric (31-month) mice. Our approach included sciatic-peroneal nerve immunolabeling coregistration, and electrophysiological recordings in a novel mouse ex-vivo preparation, the sympathetic-peroneal nerve-lumbricalis muscle (SPNL). Here, the interaction between the motoneuron and SNS at the neuromuscular junction (NMJ) and muscle innervation reflect the complexity of the living mouse. Our data show that electrical stimulation of the sympathetic neuron at the paravertebral ganglia chain enhances motoneuron synaptic vesicle release at the NMJ in young mice, while in geriatric mice, this effect is blunted. We also found that blocking β-AR prevents the sympathetic neuron from increasing NMJ transmission. Immunofluorescence coexpression analysis of immunolabeled ARs with choline acetyltransferase-, tyrosine hydroxylase-, or calcitonin gene-related peptide immunoreactive axons showed that α2B-AR is found mainly in sympathetic neurons, β1-AR in sympathetic- and motor-neurons, and both decline significantly with aging. In summary, this study unveils the molecular substrate accounting for the influence of endogenous sympathetic neurons on motoneuron-muscle transmission in young mice and its decline with aging. [ABSTRACT FROM AUTHOR]

The neuromuscular junction (NMJ) is the largest, most-complex synapse in the human body. Motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), specifically target MNs and the NMJs. However, little is known about the reasons for MN-selective neuronal and synaptic vulnerability in MN diseases. Here, utilizing a compartmental microfluidic in vitro co-culture system, we provide a possible explanation for why the NMJ, other than its unusual dimensions, differs from other synapses. By using live-imaging techniques, we discovered that cultured MNs display higher axonal and synaptic mitochondrial immobility compared with sympathetic neurons (SNs), leading to a profound enrichment of mitochondria only in the MN NMJ. Furthermore, by employing a synaptic ATP sensor, we show that mitochondrial respiration is the key contributor to ATP production in MN NMJs but not in SN synapses. Taken together, our data suggest that mitochondrial localization underlies the unique and specific qualities of MN NMJs. Our findings shed light on the role of mitochondria in MN and NMJ maintenance, and possibly indicate how mitochondria may serve as a source for selective MN vulnerability in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes.

In sympathetic neurons innervating the heart, action potentials activate voltage-gated Ca2+ channels and evoke Ca2+ entry into presynaptic terminals triggering neurotransmitter release. Binding of transmitters to specific receptors stimulates signal transduction pathways that cause changes in cardiac function. The mechanisms contributing to presynaptic Ca2+ dynamics involve regulation of endogenous Ca2+ buffers, in particular the endoplasmic reticulum, mitochondria and cyclic nucleotide targeted pathways. The purpose of this review is to summarize and highlight recent findings about Ca2+ homeostasis in cardiac sympathetic neurons and how modulation of second messengers can drive neurotransmission and affect myocyte excitability in cardiovascular disease. Moreover, we discuss the underlying mechanism of abnormal intracellular Ca2+ homeostasis and signaling in these neurons, and speculate on the role of phosphodiesterases as a therapeutic target to restore normal autonomic transmission in disease states of overactivity. [ABSTRACT FROM AUTHOR]

Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation.

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.

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. [ABSTRACT FROM AUTHOR]

Withdrawal of nerve growth factor (NGF) from sympathetic neurons causes their apoptotic death. Activation of c-Jun NH2-terminal kinase (JNK) may contribute to this death by the induction and phosphorylation of pro-apoptotic Bcl-2 proteins, such as Bax, that are involved in cytochrome c release from mitochondria and reactive oxygen species (ROS) production. Induction of either JNK or ROS may stimulate the other, and both may regulate release of apoptogenic factors from the mitochondria. In order to discern the relationship between JNK and ROS in apoptosis, we treated NGF-deprived, mouse sympathetic neurons with a JNK inhibitor and examined the effect on several important apoptotic events. Block of JNK activation prevented induction of c-Jun expression and resulted in a dose-dependent, yet surprisingly modest, increase in cell survival after 48 h of NGF deprivation. JNK suppression was also not sufficient to prevent the elevation in ROS or the release of cytochrome c from the mitochondria in NGF-deprived sympathetic neurons. Bax deletion prevents apoptotic death of NGF-deprived neurons by preventing release of cytochrome c from their mitochondria. It also prevents increased ROS on NGF deprivation. However, we found that induction of c-Jun in cells lacking Bax was equivalent to that in wild-type neurons. Our results suggest that while JNK activation plays an important role in many forms of apoptosis, it may not be a crucial regulator of Bax-dependent events involved in the apoptotic death of mouse sympathetic neurons deprived of NGF and that ROS is not involved in its activation in these cells. [ABSTRACT FROM AUTHOR]

Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed an MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor receptor 1 (TNFR1)-activated TNFα reverse signalling, in which membrane-integrated TNFα functions as a receptor for TNFR1, enhances axon growth from developing sympathetic neurons and plays a crucial role in establishing sympathetic innervation. Here, we have investigated the link between TNFα reverse signalling and axon growth in cultured sympathetic neurons. TNFR1-activated TNFα reverse signalling promotes Ca2+ influx, and highly selective T-type Ca2+ channel inhibitors, but not pharmacological inhibitors of L-type, N-type and P/Q-type Ca2+ channels, prevented enhanced axon growth. T-type Ca2+ channel-specific inhibitors eliminated Ca2+ spikes promoted by TNFα reverse signalling in axons and prevented enhanced axon growth when applied locally to axons, but not when applied to cell somata. Blocking action potential generation did not affect the effect of TNFα reverse signalling on axon growth, suggesting that propagated action potentials are not required for enhanced axon growth. TNFα reverse signalling enhanced protein kinase C (PKC) activation, and pharmacological inhibition of PKC prevented the axon growth response. These results suggest that TNFα reverse signalling promotes opening of T-type Ca2+ channels along sympathetic axons, which is required for enhanced axon growth.

The neuromuscular junction (NMJ) is indispensable for survival. This synapse between motoneurons and skeletal muscle fibers allows posture, movement and respiration. Therefore, its dysfunction creates pathologies than can be lethal. The molecular mechanisms of NMJ development and maintenance are the subject of intensive studies. This mini-review focuses on some of the most recent discoveries. An unexpected role for a protein, rapsyn, which has been known for 40 years to aggregate acetylcholine receptors has emerged. A new cell partner at NMJ has been unmasked and is challenging our understanding of the functioning of this synapse. Toxins are now used as new tools to study degeneration/regeneration. The possibility of creating human NMJ in vitro is within reach with major consequences for drug screening. Wnts are secreted neurogenic factors that have been involved in vitro in acetylcholine receptor clustering, but their precise role in vivo remains to be clarified. All these data are raising new and exciting perspectives in the field and are discussed in this Review. This is an article for the . [ABSTRACT FROM AUTHOR]

Summary Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes. [ABSTRACT FROM AUTHOR]

Bone is innervated by sympathetic and sensory neurons, which play important roles in bone remodeling. Direct neuro-osteogenic cross-talk has been demonstrated using an in vitro co-culture model comprising osteoblastic or osteoclastic cells, and neurite-spouting mouse superior cervical ganglia, suggesting that these cells are directly regulated by sympathetic neurons. The increase in sympathetic nervous activity causes bone loss through increased bone resorption and decreased bone formation, associated with β2-adrenergic activity toward both osteoblastic and osteoclastic cells. The increased bone resorption is based on the stimulation of both osteoclastogenesis and osteoclastic activity. These studies suggest β-blockers to be effective against osteoporosis, in which case there is increased sympathetic activity. Epidemiological studies have demonstrated high blood pressure to be associated with increased bone loss and β-blockers to be potential candidates for drugs to treat osteoporosis and fractures. In animal experiments, a lower dose of β-blocker also improved bone mass and biomechanical fragility in a hypertensive model. Although the interplay between sympathetic and sensory neurons is poorly understood, interestingly, sensory denervated rats have been demonstrated to show similar bone metabolism to rats with hyperactivity of sympathetic tone. This review summarized both in vitro and in vivo evidence implicating sympathetic neuronal activity in bone resorption.