Rat Superior Cervical Ganglion Research Articles

Bilirubin modulates acetylcholine receptors in rat superior cervical ganglionic neurons in a bidirectional manner.

Autonomic dysfunction as a partial contributing factor to cardiovascular instability in jaundiced patients is often associated with increased serum bilirubin levels. Whether increased serum bilirubin levels could directly inhibit sympathetic ganglion transmission by blocking neuronal nicotinic acetylcholine receptors (nAChRs) remains to be elucidated. Conventional patch-clamp recordings were used to study the effect of bilirubin on nAChRs currents from enzymatically dissociated rat superior cervical ganglia (SCG) neurons. The results showed that low concnetrations (0.5 and 2 μM) of bilirubin enhanced the peak ACh-evoked currents, while high concentrations (3 to 5.5 µM) of bilirubin suppressed the currents with an IC50 of 4 ± 0.5 μM. In addition, bilirubin decreased the extent of desensitization of nAChRs in a concentration-dependent manner. This inhibitory effect of bilirubin on nAChRs channel currents was non-competitive and voltage independent. Bilirubin partly improved the inhibitory effect of forskolin on ACh-induced currents without affecting the action of H-89. These data suggest that the dual effects of enhancement and suppression of bilirubin on nAChR function may be ascribed to the action mechanism of positive allosteric modulation and direct blockade. Thus, suppression of sympathetic ganglionic transmission through postganglionic nAChRs inhibition may partially contribute to the adverse cardiovascular effects in jaundiced patients.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Expression of nestin in superior cervical ganglia of rats is influenced by gender and gonadectomy

Neurons and glia arise from neural progenitor cells that express nestin. Although substantial changes in neuronal development were observed during the postnatal period, data concerning dynamics of nestin expression in the superior cervical ganglia (SCG) of rat during that period are lacking. It is known that gonadectomy and steroid hormones influence the development of neurons in the SCG during the postnatal period, but there are no data on how they influence the persistence of nestin expression in the SCG cells.The dynamics of nestin expression in the SCG in rats of three age groups, as well as the influence of gender and gonadectomy, was investigated.Three groups of male rats were sacrificed at 2, 3 and 6 months of age. Additional groups of male and female Sprague-Dawley rats were gonadectomized at the age of 2 months. After 30 days, they were sacrificed and SCGs were harvested and processed immunohistochemically. Immunoreactivity for nestin in the SCG was observed in satellite glia, based on their expression of s100. The proportion of neurons that were encircled with nestin-immunoreactive satellite cells (nestin encircled neurons, NEN) decreased between second and third month of age (p<0.05). The proportion of NEN was greater in the NPY+ than in the NPY− subpopulation. The proportion of NEN in the SCG of female rats was significantly higher (p<0.05) than that of both, the male rats and ovariectomised groups. The percentage of these neurons was significantly higher (p<0.05) in orchidectomised, in comparison to male rats.Results show the existence of nestin-immunoreactive satellite cells in the SCG of adult rats. A substantial decrease of nestin expression in SCG cells of rats, after the onset of sexual maturation, was observed. This decrease showed significant sex-dependence and was dramatically influenced by gonadal activity.

Sympathetic and sensory innervation of small intensely fluorescent (SIF) cells in rat superior cervical ganglion.

The sympathetic ganglion contains small intensely fluorescent (SIF) cells derived from the neural crest. We morphologically characterize SIF cells and focus on their relationship with ganglionic cells, preganglionic nerve fibers and sensory nerve endings. SIF cells stained intensely for tyrosine hydroxylase (TH), with a few cells also being immunoreactive for dopamine β-hydroxylase (DBH). Vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were distributed around some clusters of SIF cells, whereas some SIF cells closely abutted DBH-immunoreactive ganglionic cells. SIF cells contained bassoon-immunoreactive products beneath the cell membrane at the attachments and on opposite sites to the ganglionic cells. Ganglion neurons and SIF cells were immunoreactive to dopamine D2 receptors. Immunohistochemistry for P2X3 revealed ramified nerve endings with P2X3 immunoreactivity around SIF cells. Triple-labeling for P2X3, TH and VAChT allowed the classification of SIF cells into three types based on their innervation: (1) with only VAChT-immunoreactive puncta, (2) with only P2X3-immunoreactive nerve endings, (3) with both P2X3-immunoreactive nerve endings and VAChT-immunoreactive puncta. The results of retrograde tracing with fast blue dye indicated that most of these nerve endings originated from the petrosal ganglion. Thus, SIF cells in the superior cervical ganglion are innervated by preganglionic fibers and glossopharyngeal sensory nerve endings and can be classified into three types. SIF cells might modulate sympathetic activity in the superior cervical ganglion.

Expression of PTHrP and PTH/PTHrP receptor 1 in the superior cervical ganglia of rats

PTHrP and its receptor PTHR1 are found in the CNS and peripheral nervous system. The presence of PTHrP mRNA has been detected in the superior cervical ganglion (SCG), but there are no data on the cellular distribution of PTHrP and PTHR1 in the SCG. Although it is known that ovarian activity and reproductive status influence sympathetic activity, and the PTHrP/PTHR1 system is influenced by estrogens in different tissues, it is not known whether these factors have a similar effect on expression of PTHrP and PTHR1 in the nervous system. Hence, we investigated the presence and distribution of PTHrP and PTHR1 in neurons and glia of the SCG of rats, as well as the influence of ovariectomy on their expression, by using immunohistochemistry. PTHrP and PTHR1 immunoreactivity was observed in cytoplasm as well as in nuclei of almost all neurons in the SCG. In male rats, intensity of PTHrP fluorescence was significantly higher in cytoplasm of NPY−, in comparison to NPY+ neurons (p < 0.05). In female rats, 2 months post-ovariectomy, significantly lower intensity of PTHrP fluorescence in cytoplasm of the SCG neurons was observed in comparison to sham operated animals (p < 0.05). In addition to neurons, PTHrP and PTHR1 immunoreactivity was observed in most of the glia and was not influenced by ovariectomy. Results show the expression of PTHrP and its receptor, PTHR1, in the majority of neurons and glial cells in the SCG of rats. Expression of PTHrP, but not PTHR1 in the cytoplasm of SCG neurons is influenced by ovarian activity.

Fusion of Golgi-derived vesicles mediated by SNAP-25 is essential for sympathetic neuron outgrowth but relatively insensitive to botulinum neurotoxins in vitro.

Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of Mr =25000 (SNAP-25) in neurites exposed to botulinum neurotoxin typeA (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was ~1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein2 (SV2) was found to be colocalized with VAMP1-3, but not with VAMP7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.

L-type calcium channels in sympathetic α3β2-nAChR-mediated cerebral nitrergic neurogenic vasodilation.

Nicotine stimulation of α3β2-nicotinic acetylcholine receptors (α3β2-nAChRs) located on sympathetic nerves innervating basilar arteries causes calcium-dependent noradrenaline release, leading to activation of parasympathetic nitrergic nerves and dilation of basilar arteries. This study aimed to investigate the major subtype of calcium channels located on cerebral peri-vascular sympathetic nerves, which is involved in nicotine-induced α3β2-nAChR-mediated nitrergic vasodilation in basilar arteries. Nicotine- and transmural nerve stimulation (TNS)-induced dilation of isolated porcine basilar arteries was examined using in vitro tissue bath. Nicotine-induced calcium influx, nicotine-induced noradrenaline release and nicotine-induced inward currents were evaluated in rat superior cervical ganglion (SCG) neurones, peri-vascular sympathetic nerves of porcine basilar arteries and α3β2-nAChRs-expressing oocytes respectively. mRNA and protein expression of Cav 1.2 and Cav 1.3 channels were detected by RT-PCR, Western blotting and immunohistochemistry. Nicotine-induced vasodilation was not affected by ω-agatoxin TK (selective P/Q-type calcium channel blocker) or ω-conotoxin GVIA (N-type calcium channel blocker). The vasodilation, however, was inhibited by nicardipine (L-type calcium channel blocker) in concentrations which did not affect TNS-induced vasodilation, suggesting the specific blockade. Nicardipine concentration-dependently inhibited nicotine-induced calcium influx in rat SCG neurones and reduced nicotine-induced noradrenaline release from peri-vascular sympathetic nerves of porcine basilar arteries. Nicardipine (10 μm), which significantly blocked nicotine-induced vasorelaxation by 70%, did not appreciably affect nicotine-induced inward currents in α3β2-nAChRs-expressing oocytes. Furthermore, the mRNAs and proteins of Cav 1.2 and Cav 1.3 channels were expressed in porcine SCG and peri-vascular nerve terminals. The sympathetic neuronal calcium influx through L-type calcium channels is modulated by α3β2-nAChRs. This calcium influx causes noradrenaline release, initiating sympathetic-parasympathetic (axo-axonal) interaction-induced nitrergic dilation of porcine basilar arteries.

Competitive Inhibition of the Nondepolarizing Muscle Relaxant Rocuronium on Nicotinic Acetylcholine Receptor Channels in the Rat Superior Cervical Ganglia

A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

Kv2 Channel Regulation of Action Potential Repolarization and Firing Patterns in Superior Cervical Ganglion Neurons and Hippocampal CA1 Pyramidal Neurons

Kv2 family "delayed-rectifier" potassium channels are widely expressed in mammalian neurons. Kv2 channels activate relatively slowly and their contribution to action potential repolarization under physiological conditions has been unclear. We explored the function of Kv2 channels using a Kv2-selective blocker, Guangxitoxin-1E (GxTX-1E). Using acutely isolated neurons, mixed voltage-clamp and current-clamp experiments were done at 37°C to study the physiological kinetics of channel gating and action potentials. In both rat superior cervical ganglion (SCG) neurons and mouse hippocampal CA1 pyramidal neurons, 100 nm GxTX-1E produced near-saturating block of a component of current typically constituting ∼60-80% of the total delayed-rectifier current. GxTX-1E also reduced A-type potassium current (IA), but much more weakly. In SCG neurons, 100 nm GxTX-1E broadened spikes and voltage clamp experiments using action potential waveforms showed that Kv2 channels carry ∼55% of the total outward current during action potential repolarization despite activating relatively late in the spike. In CA1 neurons, 100 nm GxTX-1E broadened spikes evoked from -70 mV, but not -80 mV, likely reflecting a greater role of Kv2 when other potassium channels were partially inactivated at -70 mV. In both CA1 and SCG neurons, inhibition of Kv2 channels produced dramatic depolarization of interspike voltages during repetitive firing. In CA1 neurons and some SCG neurons, this was associated with increased initial firing frequency. In all neurons, inhibition of Kv2 channels depressed maintained firing because neurons entered depolarization block more readily. Therefore, Kv2 channels can either decrease or increase neuronal excitability depending on the time scale of excitation.

Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin

M-type potassium channels, encoded by the KCNQ family genes (KCNQ2-5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.

Excitation of rat sympathetic neurons via M1 muscarinic receptors independently of Kv7 channels

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl− concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl− channel blockers 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca2+-activated Cl− and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca2+-activated Cl− channels in addition to the well-known inhibition of Kv7 channels.

Puerarin alleviates aggravated sympathoexcitatory response induced by myocardial ischemia via regulating P2X3 receptor in rat superior cervical ganglia

Myocardial ischemia elicits a sympathoexcitatory response characterized by increase in blood pressure and sympathetic nerve activity. Puerarin, a major active ingredient extracted from the traditional Chinese plant medicine Ge-gen, has been widely used in treatment of myocardial and cerebral ischemia. However, little is known about the mechanism. Our study was aimed to explore the effect of puerarin on sympathoexcitatory response induced by myocardial ischemic injury and possible relationship with P2X3 receptor. Our results showed that puerarin alleviated systolic blood pressure and heart rate, and decreased the up-regulated of P2X3 mRNA and protein in SCG of myocardial ischemic rats. The amplitude of ATP-activated currents of SCG neurons was much larger in myocardial ischemic group than that in control group. Puerarin reduced ATP-activated currents in myocardial ischemic group and control group, and the inhibiting effects of puerarin in myocardial ischemic group were stronger than those in control group. Puerarin also significantly inhibited ATP-activated currents in HEK293 cells transfected with P2X3 receptor. These results suggest that puerarin can depress up-sympathoexcitatory response induced by myocardial ischemia via acting on P2X3 receptor in rat SCG to protect myocardium.

Differential contribution of BDNF and NGF to long-term potentiation in the superior cervical ganglion of the rat

Synaptic transmission in the sympathetic nervous system is a plastic process modulated by different factors. We characterized the effects of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) on basal transmission and ganglionic long-term potentiation (LTP) in the rat superior cervical ganglion. LTP was elicited by supramaximal tetanic stimulation (40 Hz, 3 s) of the sympathetic trunk and was quantified by measuring LTP decay time and LTP extent. Neurotrophins did not affect basal transmission, however, they differentially affected LTP. BDNF (200 ng/ml) increased LTP decay time and LTP extent 2.0-fold (p < 0.01). In contrast, NGF showed a dual effect: 200 ng/ml NGF reduced LTP decay time and LTP extent to 53% and to 32% of control value (p < 0.0001 and p < 0.02; respectively), whereas >350 ng/ml NGF significantly increased LTP decay time and LTP extent (p < 0.02). Digital analysis of compound action potentials suggests that neurotrophins could change the synchronization of unitary action potentials. Pharmacological data obtained in intact ganglia show that C2-ceramide produced a 2-fold enhancement in LTP, whereas tyrphostin AG879, an inhibitor of tyrosine kinase activity, reversed the NGF blockade and produced by itself an enhancement in LTP. In sliced ganglia we observed that an anti-TrkA antibody reversed the NGF-induced LTP blockade. Immunohistochemistry studies revealed that 83% of ganglionic neurons express TrkA, whereas 52% express p75 receptor, and 18% express TrkB receptor. We propose that p75 neurotrophin receptors and probably TrkB signaling enhance LTP, whereas TrkA signaling reduces it.

Chapter Two - Stereological and Allometric Studies on Neurons and Axo-Dendritic Synapses in Superior Cervical Ganglia

The superior cervical ganglion (SCG) plays an important role in neuropathies including Horner's syndrome, stroke, and epilepsy. While mammalian SCGs seem to share certain organizational features, they display natural differences related to the animal size and side and the complexity and synaptic coverage of their dendritic arborizations. However, apart from the rat SCG, there is little information concerning the number of SCG neurons and synapses, and the nature of relationships between body weight and the numbers and sizes of neurons and synapses remain uncertain. In the recognition of this gap in the literature, in this chapter, we reviewed the current knowledge on the SCG structure and its remodeling during postnatal development across a plethora of large mammalian species, focusing on exotic rodents and domestic animals. Instrumentally, we present stereology as a state-of-the-art 3D technology to assess the SCG 3D structure unbiasedly and suggest future research directions on this topic.

Nerve Growth Factor Sensitizes Superior Cervical Ganglion Neurons to Bradykinin

The sympathetic nervous system plays a role during chronic inflammation. Since levels of nerve growth factor (NGF) are high in inflamed tissues and, in sensory neurons, NGF controls nociception by regulating neuronal sensitivity to noxious stimuli, we hypothesize that NGF also sensitizes sympathetic neurons to bradykinin (BK), a proinflammatory peptide. We cultured rat superior cervical ganglion (SCG) neurons in the presence or absence of NGF. To assess the response of SCG neurons to Bk, we measured cytoplasmic Ca2+ elevations induced by an acute application of Bk. We found Ca2+ elevation to be increased 3-fold in the presence of NGF. These Ca2+ elevations in the presence of NGF depended on external Ca2+ and depolarization of the plasma membrane. Next, we assessed the effect of Bk on the membrane potential by electrophysiological recordings. NGF-treated neurons were significantly depolarized after Bk application, while neurons cultured in the absence of NGF showed no change in membrane potential with Bk. How does NGF render SCG neurons more responsive to a Bk stimulus? We first tested whether NGF application alters BK-induced M current inhibition. Interestingly, we observed full inhibition of M current upon application of BK with and without culture in NGF. Next, we asked whether incubation of SCG neurons with NGF alters activity of other ion channels. One of the ion channels tested, KCa1.1 (Maxi K+ channel), was reduced in current density by 50% in neurons cultured in the presence of NGF. In addition, inhibition of KCa1.1 channels with iberiotoxin in SCG neurons cultured in the absence of NGF depolarized the plasma membrane potential after Bk application. In conclusion, NGF increased Bk-induced electrical excitability of rat SCG neurons by reducing a repolarizing current mediated by KCa1.1 channels. Supported by NIH grant NS008174.

Special AT-rich sequence-binding protein-2 (Satb2) — Independent initiation of cholinergic sweat gland innervation in rat in vivo

The transcription factor special AT-rich sequence-binding protein-2 (Satb2) is induced and critical for the establishment of the cholinergic phenotype in rat superior cervical ganglion cells in culture during switching from a noradrenergic to the cholinergic phenotype upon exposure to soluble differentiation factors. To obtain evidence for Satb2 determining the sympathetic cholinergic sudomotor phenotype also in vivo, we traced the postnatal expression profile of Satb2 in the rat stellate ganglion in comparison to the vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), and the neuropeptide calcitonin gene-related peptide (CGRP), all required for sudomotor function in adult rats.

The actions of Pasteurella multocida toxin on neuronal cells

Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(1-3), Gαq, Gα11, Gα12 and Gα13 by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gαq and Gα11 could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration–inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP2 hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected.M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y2 receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y2 receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

Membrane microdomain determines the specificity of receptor-mediated modulation of Kv7/M potassium currents

The Kv7/M current is one of the major mechanisms controlling neuronal excitability, which can be modulated by activation of the G protein-coupled receptor (GPCR) via distinct signaling pathways. Membrane microdomains known as lipid rafts have been implicated in the specificity of various cell signaling pathways. The aim of this study was to understand the role of lipid rafts in the specificity of Kv7/M current modulation by activation of GPCR. Methyl-β-cyclodextrin (MβCD), often used to disrupt the integrity of lipid rafts, significantly reduced the bradykinin receptor (B2R)-induced but not muscarinic receptor (M1R)-induced inhibition of the Kv7/M current. B2R and related signaling molecules but not M1R were found in caveolin-containing raft fractions of the rat superior cervical ganglia. Furthermore, activation of B2R resulted in translocation of additional B2R into the lipid rafts, which was not observed for the activation of M1R. The increase of B2R-induced intracellular Ca2+ was also greatly reduced after MβCD treatment. Finally, B2R but not M1R was found to interact with the IP3 receptor. In conclusion, the present study implicates an important role for lipid rafts in mediating specificity for GPCR-mediated inhibition of the Kv7/M current.

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

Electrophysiological studies of upregulated P2X7 receptors in rat superior cervical ganglia after myocardial ischemic injury

Myocardial ischemic injury activates cardiac sympathetic afferent fibers and elicits a sympathoexcitatory reflex by exciting sympathetic efferent action, with resultant augmentation of myocardial oxygen consumption, leading to a vicious cycle of exaggerating myocardial ischemia. P2X7 receptor participates in the neuronal functions and the neurological disorders. This study examined the role of P2X7 receptor of superior cervical ganglia (SCG) in sympathoexcitatory reflex. Our results showed that the expression of P2X7 receptor at both mRNA and protein in SCG was increased after myocardial ischemic injury. P2X7 receptor agonists at the same concentration activated much larger amplitudes of the currents in the SCG neurons of myocardial ischemic rats than those in control rats. P2X7 receptor antagonist (brilliant blue G, BBG) significantly inhibited P2X7 receptor agonist-activated currents in the SCG neurons. Excessive phosphorylation of MAPK ERK1/2 upon the activation of P2X7 receptor might be a mechanism mediating the signal transduction after myocardial ischemic injury. Therefore, the sensitized P2X7 receptor in SCG was involved in the nociceptive transmission of sympathoexcitatory reflex induced by myocardial ischemic injury.