Rat Superior Cervical Sympathetic Ganglion Research Articles

HCN Channel Blockers Act Pre‐ and Post‐Synaptically to Inhibit Synaptic Transmission and Gain in the Rat Superior Cervical Sympathetic Ganglion

Hyperpolarization‐activated cation channels encoded by HCN subunits are permeable to Na+ and K+ ions and carry inward h‐currents. The h‐current (Ih) was originally discovered in the heart, where it is the principal pacemaker current. Ivabradine, a selective blocker of Ih, was approved in the US in 2015 as an adjunct to β‐adrenergic blockade for clinical management of heart failure. Recently we observed that sympathetic neurons in the rat superior cervical ganglion (SCG) express prominent Ih responses that can be blocked by ZD7288, another selective antagonist. The goal of the present study was to test the hypothesis that Ih modulates synaptic transmission in sympathetic ganglia. We did this using dissociated SCG neurons and using acutely isolated intact ganglia, treated with enzymes to permit whole‐cell recording, together with simultaneous presynaptic stimulation and extracellular recording. When synaptic transmission was elicited by submaximal 1 Hz stimulation in the isolated intact SCG, the extracellular postsynaptic compound action potential was inhibited by about 20% by 10–40 μM Ivabradine and by 10–20 μM ZD7288. In dissociated SCG neurons, we measured Ih under voltage clamp, fit the data to a Hodgkin‐Huxley model and then used the model to implement virtual Ih under dynamic clamp. When neurons were probed with virtual nicotinic fast EPSPs created with dynamic clamp, blocking Ih with 10–15 μM ZD7288 increased the threshold‐synaptic conductance (thresh‐gsyn) from 7.0 ± 0.9 nS to 12.1 ± 2.0 nS (p<0.0001) and decreased synaptic gain from 2.2 to 1.7 in response to noisy patterns of virtual synaptic activity that were designed to mimic physiological activity in vivo. This indicates that the native postsynaptic Ih normally enhances synaptic amplification to increase postsynaptic spike output by the SCG. When the native Ih was reconstituted with virtual Ih, synaptic amplification was restored. Shifting the voltage‐dependence of activation to mimic binding of cyclic AMP to HCN channels further modulated thresh‐gsyn and synaptic gain. To assess presynaptic actions of Ih, we then measured nicotinic cholinergic synaptic currents evoked by minimal presynaptic stimulation of the intact SCG. Ivabradine and ZD7288 both reduced peak EPSC amplitude and total charge by 40 to 50% while also increasing the failure rate. This indicates that native Ih normally enhances the probability of acetylcholine release and the amount of release by preganglionic terminals. Taken together the results suggest that clinical use of Ivabradine may have the heretofore unknown effect of reducing peripheral sympathetic activity through its pre‐ and postsynaptic effects on ganglionic transmission. This may have beneficial therapeutic consequences by countering the sympathetic hyperactivity that can drive human hypertension and that exacerbates the progression of heart failure.Support or Funding InformationSupported by the Great Rivers Affiliate of the American Heart Association through Grant‐in‐Aid 13GRNT1685007

Diabetes Impairs Synaptic Plasticity in the Superior Cervical Ganglion: Possible Role for BDNF and Oxidative Stress

The majority of diabetics develop serious disorders of the autonomic nervous system; however, there is no clear understanding on the causes of these complications. In this study, we examined the effect of streptozocin (STZ)-induced diabetes on activity-dependent synaptic plasticity, associated levels of brain-derived neurotrophic factor (BDNF) and antioxidant biomarkers in the rat sympathetic superior cervical ganglion. Diabetes (STZ-induced) was achieved by a single intraperitoneal injection of streptozocin (55mg/kg).Compound action potentials were recorded from isolated ganglia before (basal) and after repetitive stimulation, or trains of paired pulses to express ganglionic long-term potentiation (gLTP) or long-term depression (gLTD). The input/output curves of ganglia from STZ-treated animals showed a marked rightward shift along most stimulus intensities, compared to those of ganglia from control animals, indicating impaired basal synaptic transmission in ganglia from STZ-induced diabetic animals. Repetitive stimulation induced robust gLTP and gLTD in ganglia isolated from control animals; the same protocols failed to induce gLTP or gLTD in ganglia from STZ-induced diabetic animals, indicating impairment of activity-dependent synaptic plasticity in these animals. Molecular analysis revealed significant reduction in the levels of BDNF and the ratio of glutathione/oxidized glutathione. Additionally, the activity of glutathione peroxidase, glutathione reductase, catalase, and the levels of thiobarbituric acid-reactive substances were increased in ganglia from STZ-treated animals. In conclusion, impaired basal synaptic transmission and synaptic plasticity are associated with reduced BDNF and altered oxidative stress biomarkers in the sympathetic ganglia from STZ-induced diabetic animals, suggesting a possible correlation of these factors with the manifestations of STZ-induced diabetes in the peripheral nervous system.

Sympathetic neurons modulate the beat rate of pluripotent cell-derived cardiomyocytes in vitro

Although stem cell-derived cardiomyocytes have great potential for the therapy of heart failure, it is unclear whether their function after grafting can be controlled by the host sympathetic nervous system, a component of the autonomic nervous system (ANS). Here we demonstrate the formation of functional connections between rat sympathetic superior cervical ganglion (SCG) neurons and pluripotent (P19.CL6) cell-derived cardiomyocytes (P19CMs) in compartmentalized co-culture, achieved using photolithographic microfabrication techniques. Formation of synapses between sympathetic neurons and P19CMs was confirmed by immunostaining with antibodies against β-3 tubulin, synapsin I and cardiac troponin-I. Changes in the beat rate of P19CMs were triggered after electrical stimulation of the co-cultured SCG neurons, and were affected by the pulse frequency of the electrical stimulation. Such changes in the beat rate were prevented when propranolol, a β-adrenoreceptor antagonist, was added to the culture medium. These results suggest that the beat rate of differentiated cardiomyocytes can be modulated by electrical stimulation of connected sympathetic neurons.

P63 Is an Essential Proapoptotic Protein during Neural Development

The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.

Rostro-caudal variations in neuronal size reflect the topography of cellular phenotypes in the rat superior cervical sympathetic ganglion

The mammalian superior cervical ganglion (SCG) contains a complex mixture of neuronal phenotypes that selectively innervate different peripheral targets. The present study examined the rostro-caudal topography of sympathetic phenotypes in the rat SCG by analyzing the relation between cell position, size, and the expression of immunoreactivity for neuropeptide Y (NPY), calretinin, and calcitonin gene-related peptide (CGRP). We observed that 64% of SCG neurons expressed NPY and had an average diameter of ∼24 μm throughout the ganglion. Previous studies indicate that most of these cells are vasoconstrictor in function. By contrast, the size of NPY-negative neurons varied from ∼25 μm in the rostral ganglion near the internal carotid nerve to ∼30 μm in the caudal ganglion between the external carotid nerve and cervical sympathetic trunk. Many of the large NPY-negative neurons in the caudal ganglion were surrounded by dense axonal baskets that were immunoreactive for calretinin and therefore are likely to be secretomotor neurons projecting to salivary glands. Consistent with earlier reports, the rostral ganglion contained low numbers of presumptive pupillomotor neurons, based on their expression of NPY and contact with fibers containing CGRP. The present results indicate that neuronal size may provide a useful aid to cellular identification, especially in the caudal ganglion, and they provide further evidence of a topographic organization within the mammalian SCG.

Neuronal preservation in the sympathetic ganglia of rats with chronic streptozotocin-induced diabetes

Recent studies have reported that rat sympathetic neurons exposed to increased concentrations of glucose in vitro initiate an apoptotic program which culminates in neuronal cell death, a process proposed to contribute to the development of human diabetic autonomic neuropathy. We tested this hypothesis in an in vivo streptozotocin model of diabetic autonomic neuropathy using an unbiased counting method to quantitate neuron numbers in control and diabetic rats. Ten months of severe untreated diabetes failed to produce significant neuron loss in either the rat superior mesenteric or superior cervical sympathetic ganglia indicating that apoptotic neuronal cell death is unlikely to play a role in the pathogenesis of experimental diabetic autonomic neuropathy.

Vasoactive Intestinal Peptide Enhances Its Own Expression in Sympathetic Neurons after Injury

Neurons in the adult rat superior cervical sympathetic ganglion (SCG) dramatically increase their content of vasoactive intestinal peptide (VIP) and its mRNA after axotomy in vivo and after explantation. Because the VIP gene contains a functional cAMP response element, the effects of cAMP-elevating agents on VIP expression were examined. VIP, forskolin, or isoproterenol increased cAMP accumulation in explanted ganglia. Secretin, a peptide chemically related to VIP, or forskolin increased VIP levels above those seen in ganglia cultured in control medium, whereas treatment with VIP or secretin increased the level of peptide histidine isoleucine (PHI), a peptide coded for by the same mRNA that encodes VIP. VIP or forskolin also increased VIP-PHI mRNA. In contrast, isoproterenol did not alter levels of VIP, PHI, or VIP-PHI mRNA. Although VIP or forskolin increased cAMP levels in both dissociated neurons and in non-neuronal cells, isoproterenol significantly stimulated cAMP accumulation only in the latter. VIP6-28 was an effective antagonist of the actions of exogenous VIP on cAMP and VIP-PHI mRNA in neuron-enriched cultures. When adult SCG explants were cultured in defined medium, endogenous VIP immunoreactivity was released. When VIP6-28 was added to such cultures, it significantly inhibited the increase in VIP-PHI mRNA that normally occurs. These data indicate that VIP, or a closely related molecule, produced by adult neurons after injury can enhance the expression of VIP. Such a mechanism may prolong the period during which VIP is elevated after axonal damage. The possibility is also discussed that, because VIP is present in preganglionic neurons in normal animals, its release during periods of increased sympathetic nerve activity could alter VIP expression in the SCG.

Inhibition of M-current in cultured rat superior cervical ganglia by linopirdine: Mechanism of action studies

The mechanism involved in the blockade of M-current by linopirdine was studied in cultured rat superior cervical sympathetic ganglia using whole-cell patch clamp recording. The effects of modulators of intracellular signal transduction pathways on muscarine- or linopirdine-induced inhibition of M-current were compared. Intracellular addition of GDP-β-S (500 μM) attenuated M-current block by muscarine (1 μM) but not that of linopirdine (10 μM). Intracellular dialysis of GTP-γ-S (100 μM) enhanced and prolonged muscarineinduced inhibition of M-current but had no effect on the activity of linopirdine. Intracellular BAPTA (20 mM) also inhibited the effects of muscarine without affecting those of linopirdine. Intracellular application of linopirdine had no effect on either basal M-current amplitude or the ability of linopirdine to block M-current when administered extracellularly. These results indicate that M-current inhibition by linopirdine is unlikely to be either G-protein-mediated or calcium-mediated or to involve an intracellular site of action and are, therefore, consistent with a direct block of the M-channel from its extracellular side.

Effects of a cognition-enhancer, linopirdine (DuP 996), on M-type potassium currents (IK(M)) and some other voltage- and ligand-gated membrane currents in rat sympathetic neurons.

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K+ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 microM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (IK(V)), IC50 63 microM; transient (IA) current, IC50 69 microM] and produced no detectable inhibition of the fast and slow Ca(2+)-activated K+ currents IC and IAHP or of a hyperpolarization-activated cation current (IQ/Ih) at 10-30 microM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl- currents with IC50 values of 7.6 and 26 microM respectively. It is concluded that linopirdine shows some 20-fold selectivity for M-channels among different K+ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.

Interleukin‐1β increases leukemia inhibitory factor mRNA levels through transient stimulation of transcription rate

Interleukin-1β (IL-1β) induces leukemia inhibitor factor (LIF) expression in a number of cell types including non-neuronal cells of the sympathetic superior cervical ganglion (SCG). Upregulation of LIF by inflammatory cytokines is usually associated with injury response. We characterized the molecular mechanism of LIF mRNA regulation by IL-1β in explanted neonatal rat SCG and a Schwann cell line. IL-1β increases LIF mRNA levels by interacting with IL-1 receptors in SCG, since this induction could be diminished by inclusion of either soluble IL-1 receptors or IL-1 receptor antagonist. The antiinflammatory glucocorticoid dexamethasone also inhibits LIF mRNA induction by IL-1β. LIF mRNA encodes a 3′ AU-rich mRNA stability control sequence, but IL-1β does not appear to regulate the decay of LIF mRNA by this mechanism. IL-1β does not raise LIF gene transcription rate in cultured SCG 6 or 24 h after addition of IL-1β as measured by nuclear run-on assays. LIF gene transcription is induced rapidly and transiently in an immortalized Schwann cell line, returning to uninduced rates by 1 h after induction. These results suggest that the IL-1β induction of LIF gene expression is at least partially transcriptional, but that LIF mRNA increases to a greater extent than LIF transcription, suggesting the possibility of posttranscriptional regulation as well. © 1996 Wiley-Liss, Inc.

Interleukin-1 beta increases leukemia inhibitory factor mRNA levels through transient stimulation of transcription rate.

Interleukin-1 beta (IL-1 beta) induces leukemia inhibitor factor (LIF) expression in a number of cell types including non-neuronal cells of the sympathetic superior cervical ganglion (SCG). Upregulation of LIF by inflammatory cytokines is usually associated with injury response. We characterized the molecular mechanism of LIF mRNA regulation by IL-1 beta in explanted neonatal rat SCG and a Schwann cell line. IL-1 beta increases LIF mRNA levels by interacting with IL-1 receptors in SCG, since this induction could be diminished by inclusion of either soluble IL-1 receptors or IL-1 receptor antagonist. The antiinflammatory glucocorticoid dexamethasone also inhibits LIF mRNA induction by IL-1 beta. LIF mRNA encodes a 3' AU-rich mRNA stability control sequence, but IL-1 beta does not appear to regulate the decay of LIF mRNA by this mechanism. IL-1 beta does not raise LIF gene transcription rate in cultured SCG 6 or 24 h after addition of IL-1 beta as measured by nuclear run-on assays. LIF gene transcription is induced repidly and transiently in an immortalized Schwann cell line, returning to uninduced rates by 1 h after induction. These results suggest that the IL-1 beta induction of LIF gene expression is at least partially transcriptional, but that LIF mRNA increases to a greater extent than LIF transcription, suggesting the possibility of posttranscriptional regulation as well.

Autoradiographic localization of functional muscarinic receptors in the rat superior cervical sympathetic ganglion reveals an extensive distribution over non-synaptic surfaces of neuronal somata, dendrites and nerve endings.

Fast synaptic transmission in sympathetic ganglia is mediated by acetylcholine, acting on nicotinic receptors, yet muscarinic receptors are also present and are involved in the production of slow postsynaptic potentials. In order further to elucidate the role of muscarinic receptors in ganglionic transmission their distribution in the rat superior cervical sympathetic ganglion was investigated autoradiographically by use of the tritiated irreversible muscarinic ligand propylbenzilylcholine mustard. It was observed that this agent blocked the carbachol-evoked hydrolysis of inositol phospholipids in the ganglion and that this response to carbachol is itself inhibitable by selective muscarinic antagonists with a potency sequence which indicates involvement primarily of M1 receptors. Light microscope autoradiography showed that labelling inhibitable by atropine and by the M1-selective muscarinic antagonist pirenzepine was essentially confined to the margins of neuronal somata and regions of dendritic arborization, which include synaptic contacts. Quantitative electron microscope autoradiography showed that binding of the radioligand, of which approximately 70% was inhibitable by atropine and 68% by pirenzepine, was associated predominantly with surface membranes of neuronal somata, dendrites, other neurites (including axons and uncharacterized dendrites) and nerve terminal profiles, in the approximate ratios 95:85:52:45. Of the inhibitable binding over neuronal membranes in the ganglion little more than 3% was found to be synaptically located, and this involved para- or peri-synaptic regions of nerve terminal contacts rather than the specialized synaptic zone. About 5% of the inhibitable binding over neuronal membranes involved non-synaptic surfaces of nerve terminals and preterminal axon segments; almost 70% was distributed over non-synaptic surfaces of neuronal somata and dendrites, and about 21% upon other neurites. Binding sites were found not to be more highly concentrated at or adjacent to synapses than over other regions of neuronal surface membranes. About 50%, possibly more, of the binding on non-synaptic surfaces of nerve endings, and about 7% of binding upon dendritic membranes, was of non-M1, possibly M2 type, inhibitable by atropine but not by pirenzepine. Non-synaptic neuro-neuronal appositions, which involve dendrites and somata and often lie adjacent to synapses, showed rather more than twice the binding expected for each membrane individually; and neuronal membrane exposed to basal lamina lining ganglionic tissue spaces showed high levels of binding. Little inhibitable binding was seen over membranes of satellite and Schwann cells, or over cytoplasmic territories or ganglionic interstitial tissue. A model was constructed of the distribution of label, which showed that the observed results for total binding could be approximately matched by assuming the following relative densities of ligand binding sites: interstitial tissue space and supporting cells 1, soma cytoplasm 3, cytoplasm of dendrites, neurites and nerve terminals 4.5, surfaces of mesodermal elements 15, surfaces of neurites and nerve endings including sites of synapse 45, surfaces of dendrites 90, surfaces of neuronal somata 120, non-synaptic neuro-neuronal appositions 180. It is concluded that functional muscarinic receptors in this sympathetic ganglion, predominantly of the M1 type linked with slow depolarizations, but including some non-M1 receptors, are widely distributed over non-synaptic surfaces of the neuronal somata and dendrites and are not concentrated at synapses. Presynaptic autoreceptors are also present, of which half or more are of non-M1, possibly M2, type which might be inhibitory. The presence of M4 receptors is not excluded...

Induction of tissue transglutaminase in rat superior cervical sympathetic ganglia following in vitro stimulation of retinoic acid

The addition of retinoic acid (RA, 50 nM) to Dulbecco's modified Eagle's medium containing 1.0% bovine serum albumin and 50 μg/l of gentamicin markedly increased the activity of a Ca 2+-dependent tissue transglutaminase (TGase) (ca. 3.2-fold), which stabilizes newly formed protein assemblies at the sites of synapses, in isolated rat superior cervical sympathetic ganglia (SCG), which is abundant in synapses, following in vitro aerobic incubation for 3 h at 37°C. An isomer of RA, 13-cis-RA (50 nM), showed the same tendency but rather lesser magnitude (ca. 1.7-fold) in ganglionic TGase activation. Also, retinal (50 nM), a precursor of RA, had a little effect on TGase stimulation (ca. 1.5-fold) in SCG. The RA-induced enhancement of ganglionic TGase activity was completely eliminated in the presence of either actinomycin D (1.0 μg/ml), a depressant of molecular transcriptional activity, or a potent inhibitor of protein synthesis, cycloheximide (10 μg/ml). Kinetic analyses show that the stimulation of ganglionic TGase activity evoked by RA addition was associated with only an increase in V max value (ca. 3.3-fold) without change in K m value. Thus, the enzyme protein of TGase might be synthetized de novo in the ganglia in response to RA. The RA-induced activation effect of ganglionic TGase almost disappeared (ca. 1.3-fold) 1 week following denervation, by which time preganglionic cholinergic nerve terminals were degradated. In axotomized SCG, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, the RA-evoked increase in ganglionic TGase activity was attenuated (ca. 1.3-fold). These findings imply that some retinoids, especially RA effectively participate in the cholinergic potentiation of synaptic activity.

Intracellular Calcium Directly Inhibits Potassium M Channels in Excised Membrane Patches from Rat Sympathetic Neurons

Complex effects of altering intracellular [Ca 2+] on M-type K + currents have previously been reported using whole-cell current recording. To study the direct effect of Ca 2+ on M-channel activity, we have applied Ca 2+ to the inside face of membrane patches excised from rat superior cervical sympathetic ganglion cells. Ca 2+ rapidly and reversibly inhibited M-channel activity in 28/44 patches by up to 87%, with a mean IC 50 of 100 nM. This effect persisted in the absence of ATP, implying that it was not due to phosphorylation/dephosphorylation. A similar effect was observed in 13/13 cell-attached patches when cells were transiently “Ca 2+-loaded” by adding 2 mM Ca 2+ to a 25 mM K + solution bathing the extrapatch cell membrane. These observations provide new evidence that Ca 2+ can directly inhibit M channels, so supporting the view that Ca 2+ might mediate M current inhibition following muscarinic receptor activation.

LIF-and IL-1 beta-mediated increases in substance P receptor mRNA in axotomized, explanted or dissociated sympathetic ganglia.

Regulation of substance P receptor (SPR) mRNA was examined in the rat sympathetic superior cervical ganglion (SCG) in vitro and in vivo after axotomy. Interleukin-1 beta (IL-1 beta) treatment of explanted ganglia elevated levels of SPR mRNA. By contrast, dissociated cultures of purified sympathetic neurons, purified fibroblasts, and purified Schwann cells each expressed only low levels of SPR mRNA, and treatment with the cytokine did not alter levels of the receptor mRNA. Treatment of Schwann cell or fibroblast cultures with leukemia inhibitory factor (LIF) also did not alter SPR mRNA. However, treatment of pure neuronal cultures with LIF significantly elevated levels of the receptor mRNA. Further, SPR mRNA increased in pure sympathetic neurons cultured in the presence of conditioned medium from IL-1 beta treated fibroblasts or Schwann cells; this effect was blocked in the presence of LIF antibody. This suggests that the stimulatory effects of IL-1 beta on SPR mRNA in explants is mediated by LIF release. Axotomy of the SCG in vivo resulted in a significant increase in LIF mRNA. Further, axotomy resulted in a significant increase in SPR mRNA, suggesting that LIF may mediate the increase in SPR mRNA. In view of the known effects of substance P (SP) on inflammatory responses, these observations suggest that coordinated expression of SP and SPR mRNA in neurons after nerve injury may participate in inflammatory and repair processes in the ganglion.

Possible involvement of nitric oxide in carbachol-induced activation of transglutaminase in rat superior cervical sympathetic ganglia

The addition of a muscarinic agonist, carbachol (Carb, 0.1 mM), to a physiological medium markedly increased Ca 2+-dependent transglutaminase (TG) activity (approximately 10-fold) in isolated rat superior cervical sympathetic ganglia (SCG) following in vitro aerobic incubation for 30 min at 37°C. The Carb-evoked stimulation of ganglionic TG activity was considerably reduced (−51%) in the presence of N G-monomethyl- l-arginine ( l-NMMA, 50 μM), a selective inhibitor of nitric oxide (NO) synthase. While the suppressant effect of l-NMMA was completely eliminated by the addition of an excess concentration of l-arginine (0.5 mM), a precursor of NO. These observations imply that Carb-induced TG activation possibly involves NO mediation in SCG tissue. The Carb-induced elevation in ganglionic TG activity was markedly reduced (−84%) at as early as 15 min of incubation in the medium containing hemoglobin (Hb) (20 μ,M), an agent that scavenges only extracellular NO gas. Thus, it is evident that a large fraction of NO released from inside the neuronal cells to extracellular space could rapidly diffuse back into the same group of cells to induce activation of the tissue TG. Methylene blue (MB), an inhibitor of soluble guanylate cyclase (GC), at 0.5 mM, a concentration which is effective in almost abolishing the Carb-evoked synthesis of cyclic GMP (cGMP), had no effect on ganglionic TG activation induced by Carb. Therefore, an increase in cGMP synthesis mediated by NO might not participate in NO-dependent ganglionic TG activation following the stimulation with Carb. Neither actinomycin D (AMD) (1.0 μg/ml) nor cycloheximide (CHX) (10 μg/ml) caused an apparent change in Carb-evoked ganglionic TG activity. A potent inhibitor of endonuclease, aurintricarboxylic acid (ATA) (0.1 mM), led to a slight but statistically insignificant decrease (−25%) in Carb-evoked TG activation. These results suggest that the NO-mediated TG activation during synaptic transmission via muscarinic cholinergic receptors in ganglia is unlikely to be accompanied by DNA cleavage, which leads to cell degradation.

Ganglionic nicotinic acetylcholine receptor activation by the novel agonist ABT‐418

AbstractABT‐418 was functionally characterized as a neuronal nicotinic acetylcholine receptor (nAChR) channel agonist using preparations that contain nAChRs characteristic of the ganglionic subtypes. In PC12 cells, ABT‐418, like (−)nicotine, activated an inward current that decayed within seconds in the continued presence of agonist. ABT‐418 was 4‐fold less potent than (−)nicotine (EC50 = 214 ± 30 μM and 52 ± 4 μM, respectively) while the efficacy of ABT‐418 was not significantly different from (−)nicotine when the peak response amplitude was measured. Responses to 300 μM ABT‐418 were reversibly inhibited 81 ± 3% by 10 μM mecamylamine, 38 ± 1% by 10 μM dihydro‐β‐erythroidine, and 82 ± 2% by 100 μM dihydro‐β‐erythroidine. These nAChR antagonists affected the response to (−)nicotine similarly. Furthermore, responses to maximal concentrations of ABT‐418 (3 mM) and (−)nicotine (1 mM) were not additive, consistent with ABT‐418 and (−)nicotine acting through the same receptor(s). However, the Hill coefficient for ABT‐418 (1.18 ± 0.20) was smaller than that for (−)nicotine (1.77 ± 0.18), and high concentrations of ABT‐418 appeared to elicit a more rapidly decaying response than did (−)nicotine. In the rat superior cervical sympathetic ganglion also, ABT‐418 was 2.5‐fold less potent than (−)nicotine in blocking nicotinic transmission, presumably through nicotinic receptor desensitization. These studies provide the most direct evidence that ABT‐418 activates nicotinic cholinergic channels, and suggest that ABT‐418 would have reduced potency compared to (−)nicotine in peripheral ganglia, consistent with the reduced side effect liability of this novel nAChR agonist. © Wiley‐Liss, Inc.

レチノイン酸刺激によるラット上頚部交感神経節内組織トランスグルタミナーゼの誘導

レチノイン酸刺激によるラット上頚部交感神経節内組織トランスグルタミナーゼの誘導

Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy.

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 microns, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 microns), whereas VIP occurs mainly in larger neurons (26.1 microns). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed.

The intercellular communication via nitric oxide and its regulation in coupling of cyclic GMP synthesis upon stimulation of muscarinic cholinergic receptors in rat superior cervical sympathetic ganglia

Cyclic GMP (cGMP) production in rat superior cervical sympathetic ganglia (SCG) was markedly increased (ca. 7–9-fold) by the addition of either acetylcholine (ACh; 0.1 mM) or a muscarinic agonist, carbachol (Carb; 0.1 mM), in the presence of an inhibitor (3-isobutyl-1-methylxanthine) for cGMP hydrolytic enzyme during in vitro aerobic incubation at 37°C for 5 min. The ACh-induced accumulation of cGMP in SCG was effectively blocked (−73%) by the further addition of atropine (10 μM), a muscarinic antagonist, whereas a nicotinic blocker, hexamethonium (10 μM) partially antagonized (−41%) this ACh stimulation. The inhibitory effect of hexamethonium on ACh-evoked ganglionic cGMP production was effectively augmented (−83%) by addition of N G -monomethyl- l-arginine, ( l-NMMA, 50 μM), a compound that inhibits nitric oxide (NO) synthesis from l-arginine. Comparable inhibition of cGMP formation was observed following application of l-NMMA to the SCG upon stimulation of Carb. In contrast, l-NMMA had no effect on the decreased level of ACh-evoked cGMP production caused by the muscarinic antagonist. The Carb-induced elevation of ganglionic cGMP synthesis was significantly reduced within 1 min of incubation in the medium containing hemoglobin (Hb; 20 μM), an agent that scavenges only the extracellular fraction of NO. Thereafter, the tissue cGMP formation attenuated to the control level by subsequent incubation for several minutes. Addition of protein kinase C (PKC) activator,12- O-tetradecanoylphorbol 13-acetate (TPA; 1 μM) to the medium significantly decreased Carb-evoked cGMP synthesis (−61%) in SCG, whereas superoxide dismutase (SOD; 30 U/ml) only slightly suppressed the Carb stimulation. This finding supports the idea that PKC might play a role in dampening the muscarinic receptor-mediated increase in NO release within ganglionic tissue. In axotomized SCG one week prior to examination, where sympathetic neurons were degenerated and reactive proliferation of glial cells was in progress, no stimulatory effect of Carb-induced cGMP production via NO release was seen. These results provide evidence that a large fraction of NO generated upon stimulation of muscarinic receptors in sympathetic neuronal cells can possibly freely diffuse in extracellular space, and then be taken back into the same group of surrounding cells to stimulate cGMP production.