Sympathetic Ganglion Neurons Research Articles

Sympathetic Motor Neuron Dysfunction is a Missing Link in Age-Associated Sympathetic Overactivity.

Overactivity of the sympathetic nervous system is a hallmark of aging. The cellular mechanisms behind this overactivity remain poorly understood, with most attention paid to likely central nervous system components. In this work, we hypothesized that aging also affects the function of motor neurons in the peripheral sympathetic ganglia. To test this hypothesis, we compared the electrophysiological responses and ion-channel activity of neurons isolated from the superior cervical ganglia of young (12 weeks), middle-aged (64 weeks), and old (115 weeks) mice. These approaches showed that aging does impact the intrinsic properties of sympathetic motor neurons, increasing spontaneous and evoked firing responses. A reduction of M current emerged as a major contributor to age-related hyperexcitability. Thus, it is essential to consider the effect of aging on motor components of the sympathetic reflex as a crucial part of the mechanism involved in sympathetic overactivity.

Identification and In-Silico study of non-synonymous functional SNPs in the human SCN9A gene.

Single nucleotide polymorphisms are the most common form of DNA alterations at the level of a single nucleotide in the genomic sequence. Genome-wide association studies (GWAS) were carried to identify potential risk genes or genomic regions by screening for SNPs associated with disease. Recent studies have shown that SCN9A comprises the NaV1.7 subunit, Na+ channels have a gene encoding of 1988 amino acids arranged into 4 domains, all with 6 transmembrane regions, and are mainly found in dorsal root ganglion (DRG) neurons and sympathetic ganglion neurons. Multiple forms of acute hypersensitivity conditions, such as primary erythermalgia, congenital analgesia, and paroxysmal pain syndrome have been linked to polymorphisms in the SCN9A gene. Under this study, we utilized a variety of computational tools to explore out nsSNPs that are potentially damaging to heath by modifying the structure or activity of the SCN9A protein. Over 14 potentially damaging and disease-causing nsSNPs (E1889D, L1802P, F1782V, D1778N, C1370Y, V1311M, Y1248H, F1237L, M936V, I929T, V877E, D743Y, C710W, D623H) were identified by a variety of algorithms, including SNPnexus, SNAP-2, PANTHER, PhD-SNP, SNP & GO, I-Mutant, and ConSurf. Homology modeling, structure validation, and protein-ligand interactions also were performed to confirm 5 notable substitutions (L1802P, F1782V, D1778N, V1311M, and M936V). Such nsSNPs may become the center of further studies into a variety of disorders brought by SCN9A dysfunction. Using in-silico strategies for assessing SCN9A genetic variations will aid in organizing large-scale investigations and developing targeted therapeutics for disorders linked to these variations.

Changes in the expression of satellite glial cell-specific markers during postnatal development of rat sympathetic ganglia

The sympathetic ganglia represent a final motor pathway that mediates homeostatic “fight and flight” responses in the visceral organs. Satellite glial cells (SGCs) form a thin envelope close to the neuronal cell body and synapses in the sympathetic ganglia. This unique morphological feature suggests that neurons and SGCs form functional units for regulation of sympathetic output. In the present study, we addressed whether SGC-specific markers undergo age-dependent changes in the postnatal development of rat sympathetic ganglia. We found that fatty acid-binding protein 7 (FABP7) is an early SGC marker, whereas the S100B calcium-binding protein, inwardly rectifying potassium channel, Kir4.1 and small conductance calcium-activated potassium channel, SK3 are late SGC markers in the postnatal development of sympathetic ganglia. Unlike in sensory ganglia, FABP7 + SGC was barely detectable in adult sympathetic ganglia. The expression of connexin 43, a gap junction channel gradually increased with age, although it was detected in both SGCs and neurons in sympathetic ganglia. Glutamine synthetase was expressed in sensory, but not sympathetic SGCs. Unexpectedly, the sympathetic SGCs expressed a water-selective channel, aquaporin 1 instead of aquaporin 4, a pan-glial marker. However, aquaporin 1 was not detected in the SGCs encircling large neurons. Nerve injury and inflammation induced the upregulation of glial fibrillary acidic protein, suggesting that this protein is a hall marker of glial activation in the sympathetic ganglia. In conclusion, our findings provide basic information on the in vivo profiles of specific markers for identifying sympathetic SGCs at different stages of postnatal development in both healthy and diseased states.

Dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that reduces Kv7/M-current suppression and inflammatory pains.

Neuronal Kv7 voltage-gate potassium channels generate the M-current and regulate neuronal excitability. Here, we report that dehydroepiandrosterone sulfate (DHEAS) is an endogenous Kv7 channel modulator that attenuates Gq-coupled receptor induced M-current suppression. DHEAS reduced muscarinic agonist-induced Kv7-current suppression of either Kv7.1, Kv7.2, Kv7.4 or Kv7.5 homomeric currents and endogenous M-currents in rat sympathetic ganglion neurons. However, DHEAS per se did not alter the voltage-dependence of these Kv7 homomeric channels or the m1 receptor-induced activation of phospholipase C or protein kinase C. DHEAS treated Kv7.2 homomeric currents became resistant to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by voltage-activated phosphatase, Ci-VSP or eVSP. Our computational models predicted a novel binding site for DHEAS in the cytoplasmic domain of Kv7 subunits. A single point mutation of the predicted key histidine into cysteine in the rat Kv7.2 subunit, rKv7.2(H558C), resulted in a loss of effects of DHEAS on muscarinic Kv7 current suppression. Furthermore, in vivo administration of DHEAS in mice of both sexes reduced late phase pain responses in the formalin paw test. However, it did not have effects on early phase responses in the formalin paw test or responses in the hot plate test. Co-administration of a selective Kv7 inhibitor, XE991, and DHEAS eliminated analgesic effects of DHEAS in late phase responses in the formalin paw test. Collectively, these results suggest that DHEAS attenuates M-current suppression by stabilizing PIP2-Kv7 subunit interaction and can mitigate inflammatory pain.Significance StatementM-current suppression induced by stimulation of Gq-coupled receptors is a form of Kv7 current modulation that can reversibly increase neuronal excitability. This study demonstrates that DHEAS, an endogenous steroid hormone, is a novel Kv7 channel modulator that can attenuate M-current suppression without affecting basal Kv7 channel kinetics. Administration of DHEAS in vivo alleviated inflammatory pain in rodents. These results suggest that the degree of M-current suppression can be dynamically regulated by small molecules. Therefore, this novel form of Kv7 channel regulation holds promising potential as a therapeutic target for sensitized nervous activities such as inflammatory pain.

Epigenetic Up-Regulation of ADAMTS4 in Sympathetic Ganglia is Involved in the Maintenance of Neuropathic Pain Following Nerve Injury.

Sympathetic axonal sprouting into dorsal root ganglia is a major phenomenon implicated in neuropathic pain, and sympathetic ganglia blockage may relieve some intractable chronic pain in animal pain models and clinical conditions. These suggest that sympathetic ganglia participated in the maintenance of chronic pain. However, the molecular mechanism underlying sympathetic ganglia-mediated chronic pain is not clear. Here, we found that spared nerve injury treatment upregulated the expression of ADAMTS4 and AP-2α protein and mRNA in the noradrenergic neurons of sympathetic ganglia during neuropathic pain maintenance. Knockdown the ADAMTS4 or AP-2α by injecting specific retro scAAV-TH (Tyrosine Hydroxylase)-shRNA ameliorated the mechanical allodynia induced by spared nerve injury on day 21 and 28. Furthermore, chromatin immunoprecipitation and coimmunoprecipitation assays found that spared nerve injury increased the recruitment of AP-2α to the ADAMTS4 gene promoter, the interaction between AP-2α and histone acetyltransferase p300 and the histone H4 acetylation on day 28. Finally, knockdown the AP-2α reduced the acetylation of H4 on the promoter region of ADAMTS4 gene and suppressed the increase of ADAMTS4 expression induced by spared nerve injury. Together, these results suggested that the enhanced interaction between AP-2α and p300 mediated the epigenetic upregulation of ADAMTS4 in sympathetic ganglia noradrenergic neurons, which contributed to the maintenance of spared nerve injury induced neuropathic pain.

Cholinergic collaterals arising from noradrenergic sympathetic neurons in mice.

The sympathetic nervous system vitally regulates autonomic functions, including cardiac activity. Postganglionic neurons of the sympathetic chain ganglia relay signals from the central nervous system to autonomic peripheral targets. Disrupting this flow of information often dysregulates organ function and leads to poor health outcomes. Despite the importance of these sympathetic neurons, fundamental aspects of the neurocircuitry within peripheral ganglia remain poorly understood. Conventionally, simple monosynaptic cholinergic pathways from preganglionic neurons are thought to activate postganglionic sympathetic neurons. However, early studies suggested more complex neurocircuits may be present within sympathetic ganglia. The present study recorded synaptic responses in sympathetic stellate ganglia neurons following electrical activation of the pre- and postganglionic nerve trunks and used genetic strategies to assess the presence of collateral projections between postganglionic neurons of the stellate ganglia. Orthograde activation of the preganglionic nerve trunk, T-2, uncovered high jitter synaptic latencies consistent with polysynaptic connections. Pharmacological inhibition of nicotinic acetylcholine receptors with hexamethonium blocked all synaptic events. To confirm that high jitter, polysynaptic events were due to the presence of cholinergic collaterals from postganglionic neurons within the stellate ganglion, we knocked out choline acetyltransferase in adult noradrenergic neurons. This genetic knockout eliminated orthograde high jitter synaptic events and EPSCs evoked by retrograde activation. These findings suggest that cholinergic collateral projections arise from noradrenergic neurons within sympathetic ganglia. Identifying the contributions of collateral excitation to normal physiology and pathophysiology is an important area of future study and may offer novel therapeutic targets for the treatment of autonomic imbalance. KEY POINTS: Electrical stimulation of a preganglionic nerve trunk evoked fast synaptic transmission in stellate ganglion neurons with low and high jitter latencies. Retrograde stimulation of a postganglionic nerve trunk evoked direct, all-or-none action currents and delayed nicotinic EPSCs indistinguishable from orthogradely-evoked EPSCs in stellate neurons. Nicotinic acetylcholine receptor blockade prevented all spontaneous and evoked synaptic activity. Knockout of acetylcholine production in noradrenergic neurons eliminated all retrogradely-evoked EPSCs but did not change retrograde action currents, indicating that noradrenergic neurons have cholinergic collaterals connecting neurons within the stellate ganglion.

Patch-clamp analysis of nicotinic synapses whose strength straddles the firing threshold of rat sympathetic neurons.

Neurons in paravertebral sympathetic ganglia are innervated by converging nicotinic synapses of varying strength. Based upon intracellular recordings of excitatory postsynaptic potentials (EPSPs) with sharp microelectrodes these synapses were classified in the past as either primary (strong) or secondary (weak) by their ability to trigger postsynaptic action potentials. Here we present an analysis of 22 synapses whose strength straddled threshold, thereby distinguishing them from the original classification scheme for primary and secondary synapses. Recordings at 36°C were made from intact superior cervical ganglia isolated from 13 male and 3 female Sprague-Dawley rats and from 4 male spontaneously hypertensive (SHR) rats. Ganglia were pretreated with collagenase to permit patch recording. By dissecting a 1 cm length of the presynaptic cervical sympathetic nerve as part of the preparation and through use of graded presynaptic stimulation it was possible to fractionate synaptic inputs by their distinct latencies and magnitudes, and by the presynaptic stimulus threshold for each component. Comparison of cell-attached extracellular recordings with intracellular recordings of synaptic potentials and synaptic currents indicated that straddling EPSPs are not an artifact of shunting damage caused by intracellular recording. The results also showed that in cells where a single presynaptic shock elicits multiple action potentials, the response is driven by multiple synapses and not by repetitive postsynaptic firing. The conductance of straddling synapses also provides a direct estimate of the threshold synaptic conductance (9.8 nS ± 7.6 nS, n = 22, mean ± SD). The results are discussed in terms of their implications for ganglionic integration and an existing model of synaptic amplification.

Mapping and targeted viral activation of pancreatic nerves in mice reveal their roles in the regulation of glucose metabolism.

A lack of comprehensive mapping of ganglionic inputs into the pancreas and of technology for the modulation of the activity of specific pancreatic nerves has hindered the study of how they regulate metabolic processes. Here we show that the pancreas-innervating neurons in sympathetic, parasympathetic and sensory ganglia can be mapped in detail by using tissue clearing and retrograde tracing (the tracing of neural connections from the synapse to the cell body), and that genetic payloads can be delivered via intrapancreatic injection to target sites in efferent pancreatic nerves in live mice through optimized adeno-associated viruses and neural-tissue-specific promoters. We also show that, in male mice, the targeted activation of parasympathetic cholinergic intrapancreatic ganglia and neurons doubled plasma-insulin levels and improved glucose tolerance, and that tolerance was impaired by stimulating pancreas-projecting sympathetic neurons. The ability to map the peripheral ganglia innervating the pancreas and to deliver transgenes to specific pancreas-projecting neurons will facilitate the examination of ganglionic inputs and the study of the roles of pancreatic efferent innervation in glucose metabolism.

Post-Ganglionic Sympathetic Neurons can Directly Sense Raised Extracellular Na+ via SCN7a/Nax.

The relationship between dietary NaCl intake and high blood pressure is well-established, and occurs primarily through activation of the sympathetic nervous system. Nax, a Na+-sensitive Na+ channel, plays a pivotal role in driving sympathetic excitability, which is thought to originate from central regions controlling neural outflow. We investigated whether post-ganglionic sympathetic neurons from different ganglia innervating cardiac and vasculature tissue can also directly sense extracellular Na+. Using whole-cell patch clamp recordings we demonstrate that sympathetic neurons from three sympathetic ganglia (superior cervical, stellate and superior mesenteric/coeliac) respond to elevated extracellular NaCl concentration. In sympathetic stellate ganglia neurons, we established that the effect of NaCl was dose-dependent and independent of osmolarity, Cl− and membrane Ca2+ flux, and critically dependent on extracellular Na+ concentration. We show that Nax is expressed in sympathetic stellate ganglia neurons at a transcript and protein level using single-cell RNA-sequencing and immunohistochemistry respectively. Additionally, the response to NaCl was prevented by siRNA-mediated knockdown of Nax, but not by inhibition of other membrane Na+ pathways. Together, these results demonstrate that post-ganglionic sympathetic neurons are direct sensors of extracellular Na+ via Nax, which could contribute to sympathetic driven hypertension.

Sympathetic Neurons Regulate Cardiomyocyte Maturation in Culture.

Embryos devoid of autonomic innervation suffer sudden cardiac death. However, whether autonomic neurons have a role in heart development is poorly understood. To investigate if sympathetic neurons impact cardiomyocyte maturation, we co-cultured phenotypically immature cardiomyocytes derived from human induced pluripotent stem cells with mouse sympathetic ganglion neurons. We found that 1) multiple cardiac structure and ion channel genes related to cardiomyocyte maturation were up-regulated when co-cultured with sympathetic neurons; 2) sarcomere organization and connexin-43 gap junctions increased; 3) calcium imaging showed greater transient amplitudes. However, sarcomere spacing, relaxation time, and level of sarcoplasmic reticulum calcium did not show matured phenotypes. We further found that addition of endothelial and epicardial support cells did not enhance maturation to a greater extent beyond sympathetic neurons, while administration of isoproterenol alone was insufficient to induce changes in gene expression. These results demonstrate that sympathetic neurons have a significant and complex role in regulating cardiomyocyte development.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Emerging importance of satellite glia in nervous system function and dysfunction.

Satellite glial cells (SGCs) closely envelop cell bodies of neurons in sensory, sympathetic and parasympathetic ganglia. This unique organization is not found elsewhere in the nervous system. SGCs in sensory ganglia are activated by numerous types of nerve injury and inflammation. The activation includes upregulation of glial fibrillary acidic protein, stronger gap junction-mediated SGC–SGC and neuron–SGC coupling, increased sensitivity to ATP, downregulation of Kir4.1 potassium channels and increased cytokine synthesis and release. There is evidence that these changes in SGCs contribute to chronic pain by augmenting neuronal activity and that these changes are consistent in various rodent pain models and likely also in human pain. Therefore, understanding these changes and the resulting abnormal interactions of SGCs with sensory neurons could provide a mechanistic approach that might be exploited therapeutically in alleviation and prevention of pain. We describe how SGCs are altered in rodent models of four common types of pain: systemic inflammation (sickness behaviour), post-surgical pain, diabetic neuropathic pain and post-herpetic pain.

Transsynaptic connections between the hypothalamus and adipose tissue: relationship to reproduction

Neurophysiological mechanisms that control energy balance are reciprocally linked to those that control reproduction. Neuromorphological studies using retrograde tracing methods revealed that nerve cells within the central (CNS) and autonomic (ANS) nervous systems in different species, including the pig, are transsynaptically connected to different fat tissue depots. In the pig, neurons localised in the paraventricular nucleus, supraoptic nucleus and arcuate nucleus were infected with pseudorabies virus (PRV) 9 days after injections into both the perirenal and subcutaneous adipose tissue depots. Infected neurons were in the ventromedial nucleus, dorsomedial nucleus and preoptic area after injection of PRV into perirenal adipose tissue, while infected cells in the lateral hypothalamic area projected only to the subcutaneous adipose tissue depot. Additionally, numerous centres of the ANS innervate adipose tissue depots in the pig. Fast blue stained (FB+) neurons, which projected to the subcutaneous adipose tissue overlaying the thoracolumbar area were located in the thoraco-lumbar region of the sympathetic chain ganglia (SChG). However, neurons supplying perirenal and mesentery adipose tissue depots were found in both the SChG and prevertebral ganglia. The vast majority of labelled neurons, in both the CNS and ANS, which innervated adipose tissue depots, expressed leptin receptor (OBR) immunoreactivity. The purpose of this brief review is to establish evidence for a multisynaptic circuit of neurons, which innervate adipose tissue in the pig and demonstrate that hypothalamic nuclei and sympathetic ganglion neurons involved in reproductive processes are transsynaptically connected to different adipose tissue depots.

Ca2+-independent but voltage-dependent quantal catecholamine secretion (CiVDS) in the mammalian sympathetic nervous system

Action potential-induced vesicular exocytosis is considered exclusively Ca2+ dependent in Katz's Ca2+ hypothesis on synaptic transmission. This long-standing concept gets an exception following the discovery of Ca2+-independent but voltage-dependent secretion (CiVDS) and its molecular mechanisms in dorsal root ganglion sensory neurons. However, whether CiVDS presents only in sensory cells remains elusive. Here, by combining multiple independent recordings, we report that [1] CiVDS robustly presents in the sympathetic nervous system, including sympathetic superior cervical ganglion neurons and slice adrenal chromaffin cells, [2] uses voltage sensors of Ca2+ channels (N-type and novel L-type), and [3] contributes to catecholamine release in both homeostatic and fight-or-flight like states; [4] CiVDS-mediated catecholamine release is faster than that of Ca2+-dependent secretion at the quantal level and [5] increases Ca2+ currents and contractility of cardiac myocytes. Together, CiVDS presents in the sympathetic nervous system with potential physiological functions, including cardiac muscle contractility.

Exosomes derived from cardiac parasympathetic ganglionic neurons inhibit apoptosis in hyperglycemic cardiomyoblasts.

Diabetic cardiomyopathy is known to involve two forms of cardiac cell death: apoptosis and necrosis. However, it remains unknown whether hyperglycemia-induced apoptosis in the H9c2 cell culture system is inhibited by parasympathetic ganglionic neurons (PGN) derived exosomes (exos). We isolated PGN and sympathetic ganglionic neurons (SGN) from the right stellate ganglion in rats, and derived exos from these sources. H9c2 cells were divided into 4 groups: (1) Control, (2) H9c2 + Glucose (100mmol/L), (3) H9c2 + Glucose + PGN-exos, and (4) H9c2 + Glucose + SGN-exos. We determined cell proliferation and viability with an MTT assay kit, and assessed apoptotic cell death with TUNEL staining and ELISA. Data were further confirmed by analyzing the presence of pro-apoptotic proteins Caspase-3 and Bax, and anti-apoptotic protein Bcl-2. Glucose exposed H9c2 cells significantly reduced cell viability, which was improved by PGN-exos, but not by SGN-exos. Furthermore, increased apoptosis in hyperglycemia in H9c2 cells was confirmed with TUNEL staining and cell death ELISA which demonstrated significantly (p < 0.05) reduction with PGN-exos treatment, but not with SGN-exos. Moreover, high expression of pro-apoptotic proteins Caspase-3 and Bax was reduced following treatment with PGN-exos; however, SGN-exos were unable to reduce the expression. Significantly reduced anti-apoptotic protein Bcl-2 following glucose treatment was improved with PGN-exos. Therefore, our data suggest that hyperglycemia induces apoptosis in H9c2 cells and decreases cell viability, and that PGN-exos are able to inhibit apoptosis, improve cell viability, and restore levels of anti-apoptotic protein Bcl-2.

The effects of sympathetic nerve damage on satellite glial cells in the mouse superior cervical ganglion.

Neurons in sensory, sympathetic, and parasympathetic ganglia are surrounded by satellite glial cell (SGCs). There is little information on the effects of nerve damage on SGCs in autonomic ganglia. We studied the consequences of damage to sympathetic nerve terminals by 6-hydroxydopamine (6-OHDA) on SGCs in the mouse superior cervical ganglia (Sup-CG). Immunostaining revealed that at 1-30 d post-6-OHDA injection, SGCs in Sup-CG were activated, as assayed by upregulation of glial fibrillary acidic protein. Intracellular labeling showed that dye coupling between SGCs around different neurons increased 4-6-fold 1-14 d after 6-OHDA injection. Behavioral testing 1-7 d post-6-OHDA showed that withdrawal threshold to tactile stimulation of the hind paws was reduced by 65-85%, consistent with hypersensitivity. A single intraperitoneal injection of the gap junction blocker carbenoxolone restored normal tactile thresholds in 6-OHDA-treated mice, suggesting a contribution of SGC gap junctions to pain. Using calcium imaging we found that after 6-OHDA treatment responses of SGCs to ATP were increased by about 30% compared with controls, but responses to ACh were reduced by 48%. The same experiments for SGCs in trigeminal ganglia from 6-OHDA injected mice showed no difference from controls, confirming that 6-OHDA acted selectively on sympathetic nerves. However, systemic inflammation induced by lipopolysaccharide did not affect SGCs of Sup-CG, but did influence SGCs in trigeminal ganglia in the same manner as 6-OHDA did on SGCs in Sup-CG. We conclude that even though SGCs in sympathetic and sensory ganglia are morphologically similar, they are quite different functionally, particularly after damage.

Nicotine is neuroprotective to neonatal neurons of sympathetic ganglion in rat.

Sympathetic neurons of SCG are dependent on availability of nerve growth factor (NGF) for their survival. SCG neurons express nicotinic receptors (nAChR) whose expression levels are modulated by nicotine. Nicotine exerts multiple effects on neurons, including neuroprotection, through nAChR binding. Although sympathetic neurons express robust levels of nAChR, a possible neuroprotective role for nicotine in these neurons is not well-understood. Therefore we determined the effect of nicotine exposure on survival of SCG neurons during NGF withdrawal in a well-established cell culture system. NGF was withdrawn in rat neonatal SCG neuron cultures which were then treated with either 10 μM nicotine alone or with nAChR antagonists 0.1 μM α-bungarotoxin (antagonist for α7 subunit bearing nAChR) and 10 μM mecamylamine (non-specific antagonist for ganglionic nAChR) for 48 h. Apoptotic death was determined by TUNEL staining. Cell survival was also determined by MTS assay. Western blot analysis of ERK1/2 was also performed. Our results showed that exposure to 10 μM nicotine significantly reduced apoptotic cell death in SCG neurons resulting from NGF withdrawal as shown by fewer TUNEL positive cells. The MTS assay results also revealed that 10 μM nicotine concentration significantly increased cell survival thus indicating neuroprotective effect of nicotine against cell death resulting from NGF withdrawal. Nicotinic receptor antagonists (bungarotoxin & mecamylamine) attenuated the effect of nicotine's action of neuroprotection. Western blot analysis showed an increased expression of ERK1/2 in nicotine treated cultures suggesting nicotine provided neuroprotection in SCG neurons by increasing the expression of ERK1/2 through nicotinic receptor dependent mechanisms.

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.

Synaptic Plasticity in Cardiac Innervation and Its Potential Role in Atrial Fibrillation

Synaptic plasticity is defined as the ability of synapses to change their strength of transmission. Plasticity of synaptic connections in the brain is a major focus of neuroscience research, as it is the primary mechanism underpinning learning and memory. Beyond the brain however, plasticity in peripheral neurons is less well understood, particularly in the neurons innervating the heart. The atria receive rich innervation from the autonomic branch of the peripheral nervous system. Sympathetic neurons are clustered in stellate and cervical ganglia alongside the spinal cord and extend fibers to the heart directly innervating the myocardium. These neurons are major drivers of hyperactive sympathetic activity observed in heart disease, ventricular arrhythmias, and sudden cardiac death. Both pre- and postsynaptic changes have been observed to occur at synapses formed by sympathetic ganglion neurons, suggesting that plasticity at sympathetic neuro-cardiac synapses is a major contributor to arrhythmias. Less is known about the plasticity in parasympathetic neurons located in clusters on the heart surface. These neuronal clusters, termed ganglionated plexi, or “little brains,” can independently modulate neural control of the heart and stimulation that enhances their excitability can induce arrhythmia such as atrial fibrillation. The ability of these neurons to alter parasympathetic activity suggests that plasticity may indeed occur at the synapses formed on and by ganglionated plexi neurons. Such changes may not only fine-tune autonomic innervation of the heart, but could also be a source of maladaptive plasticity during atrial fibrillation.

Expression and regulation of M-type K+ channel in PC12 cells and rat adrenal medullary cells.

M-type K+ channels contribute to the resting membrane potential in the sympathetic ganglion neurons of various animals, whereas their expression in adrenal medullary (AM) cells has been controversial. The present experiment aims to explore the expression of M channels comprising the KCNQ2 subunit in the rat AM cell and its immortalized cell line PC12 cells at the protein level and how its expression in PC12 cells is regulated. The KCNQ2 isoform was recognized in homogenates of PC12 cells but not the rat adrenal medullae by immunoblotting and KCNQ2-like immunoreactivity (IR) was detected in PC12 cells but not in rat AM cells. When the PC12 cells were maintained in a dexamethasone-containing medium, KCNQ2-like IR in the cells was suppressed, whereas the removal of fetal bovine serum from the culture medium for 1day resulted in an increase in KCNQ2-like IR. A similar enhancement occurred when PC12 cells were cultured under conditions where glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) activities were suppressed. These morphological findings were confirmed in functional analysis. The cells cultured in the presence of an inhibitor of either GR or MR exhibited larger amplitudes of Ca2+ signal in response to an M channel inhibitor than did the cells in its absence, whereas the resting Ca2+ level in the former was lower than that in the latter. These results indicate that the M channel is not expressed in rat AM cells and this absence of expression may be ascribed to the suppression by glucocorticoid activity.