Superior Cervical Ganglion Of Cat Research Articles

Benzodiazepines decrease the release of [ 3H]noradrenaline and of [ 3H]acetylcholine in the cat superior cervical ganglion

The effects of two benzodiazepines, diazepam and clonazepam, were studied on the release of [ 3H]noradrenaline and of [ 3H]acetylcholine elicited by preganglionic stimulation of the cat isolated superior cervical ganglion and on the contractile responses evoked by either nerve stimulation or exogenous noradrenaline in the cat isolated nictitating membrane. Both 10 μM diazepam and 10 μM clonazepam reduced by approximately 50% the release of [ 3H]noradrenaline and of [ 3H]acetylcholine in the cat ganglion whereas they did not modify the contractile responses in the nictitating membrane. It is concluded that benzodiazepines are also peripheral neuroactive agents and that they exhibit a tissue-selective action within the same animal species.

Maintenance by glycyl-L-glutamine in vivo of molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

The 24-hr intracarotid infusion of plasma-treated glycyl-L-glutamine (3 microM) produced significant enhancement of the monomeric G1 and tetrameric G4 forms of acetylcholinesterase of the cat superior cervical ganglion 48 hr after denervation, in comparison with denervated, noninfused controls. No significant effect of glycyl-L-glutamine could be demonstrated 4 or 6 days after denervation. These findings are consistent with the conclusion, drawn from a previous in vitro study, that glycyl-L-glutamine acts at a stage prior to the aggregation of the G1 form into higher polymers to maintain the acetylcholinesterase content of denervated ganglia. It is proposed that the dipeptide may regulate the transcription of the DNA for acetylcholinesterase to its corresponding mRNA.

Antihistaminic activity of stobadine in the peripheral and central nervous system.

Stobadine in low doses induced hyperpolarization, and in high doses depolarization and inhibition of transmission in the cat superior cervical ganglion. The ganglionic depolarizing action of histamine was inhibited by stobadine in low doses. Stobadine partially depressed ganglionic responses to isoprenaline, serotonin, M1 muscarinic agonist McN-A-343 and clonidine only in high doses. Stobadine administered into the third cerebral ventricle of anaesthetized cats evoked dose-dependent hypotension without changes in heart rate. Intracerebroventricular (i.c.v.) administration of histamine evoked hypertension followed by hypotension. Both effects were inhibited by i.c.v. pretreatment with stobadine. These results indicate that in nervous tissue stobadine antagonises histamine responses presumably due to H1-receptor stimulation.

The actions of histamine in cat and rabbit superior cervical ganglia.

Depolarization of the cat superior cervical ganglion (SCG) evoked by histamine was antagonized by mepyramine. Histamine-induced depolarization, indicated by changes in the negative and positive afterpotentials of ganglionic action potentials, was decreased by pretreatment with aminophylline. In the cat SCG, histamine evoked stimulus bound decremental oscillatory potentials (SBDOP). In the rabbit SCG, only depolarization and no SBDOP were observed after the administration of histamine. Histamine-induced SBDOP were increased by isoprenaline and decreased by Leu-enkephalin pretreatment. Our results indicate that histamine evokes depolarization and an increase in excitability of the cat SCG which seems to be mediated by H1-receptors.

Effects of glycyl-L-glutamine in vitro on the molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Normal and preganglionically denervated cat superior cervical ganglia were sectioned and cultured for 24 or 48 hr, with or without preliminary inactivation of acetylcholinesterase, and in the presence or absence of 10(-5) M glycyl-L-glutamine. They were then homogenized, and the molecular forms of acetylcholinesterase were analyzed by sucrose gradient sedimentation. We observed an increased proportion of the globular monomeric G1 form, and to a lesser extent of the dimeric G2 and tetrameric membranous G4 forms, of acetylcholinesterase in the glycyl-L-glutamine-treated compared with the control cultures. There was only a small increase in the total acetylcholinesterase activity and no significant variation in the activity of the metabolic enzyme lactate dehydrogenase. It therefore seems likely that glycyl-L-glutamine, or the endogenous neurotrophic factor, maintains acetylcholinesterase in the preganglionically denervated ganglia in vivo by specifically increasing the biosynthesis of the monomeric G1 form, but not that of other proteins; these trophic factors do not seem to promote the polymerization of G1 into the more complex G2 and G4 forms.

Acetylcholine mobilization in a sympathetic ganglion in the presence and absence of 2-(4-phenylpiperidino)cyclohexanol (AH5183).

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.

Direct neurotrophic action of glycyl-L-glutamine in the maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Intracarotid infusion of glycyl-L-glutamine (Gly-Gln) was shown previously to oppose the fall in the acetylcholinesterase and butyrylcholinesterase contents of the cat superior cervical ganglion (SCG) that otherwise follows preganglionic denervation. However, its effect was demonstrable only on the vascularly remote left SCG but not on the directly infused right SCG. Accordingly, it was concluded that a metabolite of Gly-Gln, formed in the blood, is an active neurotrophic factor. Glycyl-L-glutamic acid and L-glutamic acid were subsequently found to have a similar but less marked effect on both SCG. In the present study an alternative explanation has been tested: that Gly-Gln must combine slowly with some component of plasma to enable it to penetrate the ganglion cells and exert its neurotrophic effect. Findings are consistent with the latter proposal.

Do presynaptic opiate receptors and α-adrenoceptors alter acetylcholine release from a sympathetic ganglion by a similar mechanism?

The present experiments tested the possible involvement of a calcium-sensitive mechanism in the α-adrenoceptor-and opiate receptor-mediated inhibition of acetylcholine release from the cat superior cervical ganglion. First, the calcium-dependence of evoked acetylcholine release was measured in the presence and absence of the α-adrenoceptor agonist noradrenaline or of the opiate receptor agonist [Met 5]enkephalin-Arg 6-Phe 7. When ganglia were perfused with Krebs medium containing [Ca 2+] = 2.4, 1.2, 0.6, 0.2 mM, evoked release of acetylcholine was depressed by both agonists and the inhibition increased with reduced levels of extracellular Ca 2+; this was especially evident when calcium in the medium was reduced to 0.2 mM. Second, the effects of both noradrenaline and [Met 5]enkephalin-Arg 6-Phe 7 on calcium influx into presynaptic nerve endings was determined by measuring the accumulation of 45Ca into ganglia in the presence and absence of either drug. Both agonists reduced the stimulation-induced increase in 45Ca accumulation. The effect of noradrenaline to reduce calcium influx was blocked by yohimbine or by phentolamine; the effect of [Met 5]enkephalin-Arg 6-Phe 7 to decrease 45Ca accumulation by ganglia was blocked by naloxone. It is concluded that activation of presynaptic opiate receptors and α-adrenoceptors in the cat superior cervical ganglion can alter acetylcholine release by a similar mechanism, i.e. to reduce Ca 2+ influx during preganglionic nerve stimulation.

Effect of endogenous opioid peptides on acetylcholine release from the cat superior cervical ganglion: selective effect of a heptapeptide

The present experiments show the presence of both metenkephalin-like and met-enkephalin-Arg6-Phe7-like immunoreactivity in the superior cervical ganglion of the cat; this was determined by radioimmunoassay after high-pressure liquid chromatography separation of tissue extracts. There was measurable efflux of both peptides, as determined by radioimmunoassay of ganglionic perfusates; this measure was increased by thiorphan, an enkephalinase inhibitor. The effect of the 2 peptides on ACh release was determined: The stable analog of methionine-enkephalin, D-Ala2-methionine-enkephalinamide, did not affect ACh release from the ganglion; in contrast, methionine-enkephalin-Arg6-Phe7 significantly depressed evoked ACh release. The effect of met-enkephalin-Arg6-Phe7 to decrease ACh release was antagonized, although only partially, by the opioid antagonist naloxone. Thus, it appears that methionine-enkephalin-Arg6-Phe7 alters ACh release from the superior cervical ganglion by acting, at least in part, on a presynaptic opioid receptor. The results suggest that in the cat superior cervical ganglion, the heptapeptide enkephalin might have a significant role in the regulation of synaptic transmission, which is unrelated to its potential function as a precursor for methionine-enkephalin.

Time course and frequency dependence of synaptic vesicle depletion and recovery in electrically stimulated sympathetic ganglia.

The mammalian superior cervical sympathetic ganglion has been extensively used to study the kinetics of ACh metabolism and release. The present investigation examined the time course of changes in the number of synaptic vesicles and abundance of plasma membrane at preganglionic nerve terminals using stimulation protocols similar to those used in previous biochemical and electrophysiological studies. Continuous stimulation of the preganglionic trunk to the cat superior cervical ganglion in vivo produced an initially rapid fall in the number of clear synaptic vesicles followed by a subsequent plateau. Reciprocal changes in plasma membrane occurred with a similar time course. The plateau phase is interpreted as a steady-state where vesicle exocytosis is balanced by the rate of vesicle reformation from plasma membrane. During quiescent recovery, restoration of normal resting ultrastructure is initially rapid but slows with time as vesicle number and plasma membrane abundance approach pre-stimulation values, indicating that the rate of vesicle reformation at the end of stimulation is high and proportional to the number of vesicles incorporated into the plasma membrane. These results are interpreted as consistent with the 'vesicle hypothesis' of neurotransmitter release.

Vasoactive intestinal polypeptide facilitates the late component of the 5-hydroxytryptamine-induced discharge in the cat superior cervical ganglion

The intra-arterial administration of vasoactive intestinal polypeptide (VIP, 1–10 μg, i.a.) to the cat superior cervical ganglion facilitated or unmasked the late component but not the early component of the 5-hydroxytryptamine (5-HT, 0.5–50 μg, i.a.)-induced postganglionic discharge. The facilitation occurred in acutely and chronically decentralized ganglia. The early and late 5-HT discharges were blocked by MDL-72222, a 5-HT antagonist, but not by cholinergic antagonists. These data together with previous observations indicate that VIP selectively facilitates slow cholinergic and non-cholinergic excitatory mechanisms in autonomic ganglia.

Vasoactive intestinal polypeptide elecits a discharge in chronically decentralized sympathetic ganglia

Vasoactive intestinal polypeptide (VIP, 5–50 μg) injected intraarterially to the chronically decentralized cat superior cervical ganglion elicited a prolonged (2–5 min) postganlionic discharge which was resistant to cholinergic blocking agents but was blocked by [Leu 5enkephalin and GABA (10–200 μg i.a.). VIP did not elecit a discharge in acutely decentralized ganglia. These findings indicate that VIP has direct excitatory effects on ganglion cells and that these excitatory effects are enhanced following degeneration of the preganglionic nerve terminals.

Formation of synaptic vesicles in the superior cervical ganglion of cat: choline dependency.

De novo formation of synaptic vesicles was studied electron microscopically in synapses of the superior cervical ganglion of cat. Following prolonged electrical stimulation in a choline deficient condition, almost complete loss of vesicle content was observed. After choline administration, together with the partial restoration of transmission, the reappearance of vesicles could be seen in nerve terminals. In interpreting our observations, the double role of choline (transmitter precursor and membrane constituent) is emphasized.

Evidence that endogenous catecholamines can regulate acetylcholine release in a sympathetic ganglion

The objective of this study was to test whether activation of α-adrenoceptors by endogenously released catecholamines alters the release of acetylcholine (ACh) from the cat superior cervical ganglion. The α-adrenoceptor agonists noradrenaline and clonidine depressed evoked ACh release; this effect was concentration-dependent; it was apparent during preganglionic stimulation at 20 Hz, but not so at lower frequencies of stimulation. The inhibitory effect of noradrenaline on evoked ACh release was reversed by yohimbine, by phentolamine and, to a lesser extent, by prazosin. Thus, exogenous amines can depress evoked ACh release by an action on presynaptic α-adrenoceptors. To determine if activation of these receptors by endogenous amines inhibits ACh release, we tested whether the α-adrenoceptor antagonists enhance ACh release. Yohimbine and phentolamine increased evoked ACh release during preganglionic stimulation at 20 Hz, but not during stimulation at 5 Hz, suggesting that endogenous, like exogenous, amine can depress evoked ACh release from preganglionic nerve terminals.

In vitro metabolism of [ 3H]NA induced by pargyline in the superior cervical ganglion

1. 1. The deaminated glycol, [ 3H]DOPEG, is the main metabolite in spontaneous outflow from ganglia labelled with [ 3H]NA. 2. 2. After exposure to pargyline 0.5 mM, [ 3H]NMN formation may be the first step of the metabolism of [ 3H]NA in the cat superior cervical ganglion.

Horseradish peroxidase uptake of the synaptic vesicles in the superior cervical ganglion of the cat

The function-dependent changes in synaptic vesicle number and horseradish peroxidase (HRP) labeling were studied in the synapses of cat superior cervical ganglia. It was found that only a low percentage of vesicles contained the extracellular marker. Supramaximal stimulation (15 min) and subsequent rest (5 min) did not result in significant changes in vesicle number and the percentage of labeled vesicles also remained at about the same level. The low and activity-independent HRP labeling rate does not exclude the possibility of vesicle recycling but indicates that it might be only indirectly connected to the transmitter release itself.

Activation of acetylcholine synthesis in cat sympathetic ganglia: dependence on external choline and sodium‐pump rate.

Acetylcholine synthesis in the perfused cat superior cervical ganglion is maximally activated without activation of release during a 10 min recovery in Locke solution following a 15 min period of Na-pump inhibition by perfusion with K-free Locke solution; choline (5 X 10(-5) M) being present throughout. This procedure combined with the use of very high rates of perfusion flow has now permitted an examination of the roles of choline uptake and Na in the activation of synthesis. The data were analysed by analysis of variance as a basis for assessing experimental error and by Bartlett's test to assess equality of variance. Significance of differences between groups was estimated from this analysis (see Appendix). By selective omission of choline, either with or without addition of hemicholinium-3 (HC-3), in the K-free or in the recovery period it was found that choline is only taken up for formation of acetylcholine in the recovery period. With the use of different concentrations of choline in the recovery period, and omission of choline in the K-free period, it was found that the rate of acetylcholine synthesis increased with increasing choline concentration in conformity with Michaelis-Menten kinetics. The choline concentration giving half-maximal synthesis rate was 3.6 microM. Addition of 10(-6) M-HC-3 during recovery completely abolished synthesis in the presence of 5 X 10(-6) M-choline, and 2.5 X 10(-7) M-HC-3 reduced it by 68%. These values for choline dependence and inhibitory potency of HC-3 are similar to those found for high affinity choline transport in brain synaptosomes, indicating that the same system operates in brain and in ganglia. In additional experiments in which choline was omitted in the K-free period and with 5 X 10(-5) M-choline in the recovery fluid a reduction of external Na to 50 mM during recovery did not reduce significantly the maximal rate of acetylcholine synthesis. Further reduction to 25 nM, which would be expected to abolish the Na gradient, reduced the rate of synthesis by only 18%. The presence of 2 X 10(-5) M-ouabain during recovery in normal Locke solution containing 5 X 10(-5) M-choline abolished synthesis. It is concluded that choline uptake for acetylcholine synthesis in ganglia is via the high affinity transporter; that the transport is rate limiting for acetylcholine synthesis and; that the transport process is intimately linked to Na-pump rate.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycyl-L-glutamine, a precursor, and glycyl-L-glutamic acid, a neurotrophic factor for maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat in vivo.

L. W. Haynes and M. E. Smith have reported [(1985) Biochem. Soc. Trans. 13, 174-175] that glycyl-L-glutamine (Gly-Gln) increases the A12 and G4 forms of acetylcholinesterase (AcChoEase) in cultured embryonic rat skeletal muscle. Since Gly-Gln meets the criteria established for the neurotrophic factor (NF) in extracts of central nervous system/sciatic nerves that maintains AcChoEase and butyrylcholinesterase (BtChoEase) in the denervated cat superior cervical ganglion (SCG) in vivo, it was tested by the latter procedure. Solutions of Gly-Gln (10(-7)-10(-3) M) in 0.9% NaCl solution were infused for 24 hr via the right common carotid artery of cats with preganglionically denervated SCG, following ligation of the external carotid and lingual arteries. At 48 hr postdenervation, the AcChoEase and BtChoEase contents of the right SCG were within the range of similarly treated controls infused with 0.9% NaCl solution; the AcChoEase and BtChoEase contents of the left SCG, where the infused solutions arrived by way of a much more circuitous route, were significantly elevated at concentrations of Gly-Gln of 10(-5) M and higher. This suggested that the neurotrophic effect on the left SCG was produced by a metabolite of Gly-Gln. Accordingly, glycine, L-glutamine, and glycyl-L-glutamic acid (Gly-Glu) were then tested. Glycine and L-glutamine were inactive; Gly-Glu, 10(-6)-10(-5) M, exerted a significantly positive neurotrophic effect at both the right and left SCG; at 10(-4) M, the effect was absent. The method employed currently for preparation of extracts of SCG for assay of AcChoEase, BtChoEase, and protein contents (homogenization of scissor-minced ganglia in water) was compared with homogenization in molar NaCl/1% Triton X-100. Values obtained by the former procedure, in comparison with the latter, were 91% +/- 7% for AcChoEase and 83% +/- 7% for BtChoEase, expressed as substrate hydrolyzed per mg of protein per min.

Morphine decreases 3H-norepinephrine release and increases endogenous norepinephrine levels in the isolated cat superior cervical ganglion

The isolated cat superior cervical ganglion (SCG) was labeled in vitro with either 3H-norepinephrine ( 3HNE) or 3H-choline and stimulated through its preganglionic trunk. The release of 3HNE and of 3H-acetylcholine ( 3HACh) elicited by the stimulation was measured under control conditions and in the presence of drugs. The incubation during 30 min with 10 μM morphine lead to a 70 % decrease in the amount of 3HNE released in response to the preganglionic stimulation (10 Hz, 80 V, during 5 min). No further decrease in 3HNE release was produced by a 10 times higher concentration of morphine. The reduction in 3HNE release caused by morphine was coincident with a 60 % increase in the endogenous content of NE. Both effects of morphine were entirely prevented by an antagonist of opioid receptors, 1.0 μM naltrexone. The opioid antagonist did not modify by itself either the stimulation-induced release of 3HNE or the endogenous content of NE. The basal efflux of 3HNE was not altered by morphine. In ganglia labeled with 3H-choline, morphine (10 and 100 μM) did not modify either the basal efflux of 3HACh or the release of 3HACh evoked by stimulation of the preganglionic trunk (5 Hz, 40 V, during 5 min). These observations suggest that in the cat SCG morphine has a direct action on the dendrites of the postganglionic neuron which store and release NE. The effects of morphine in vitro on 3HNE release and on the tissue levels of NE may be mediated through the interaction with dendritic opioid receptors.

Serotonin release from the superior cervical ganglion of the cat

Serotonin of the superior cervical ganglion is contained in a distinct and separate population of small intensely fluorescent (SIF) cells. We provide evidence, in in vitro experiments, that newly synthesized serotonin can be released in the cat superior cervical ganglion from the serotonin-containing SIF cells. Resting steady state in the release of [ 3H]serotonin was observed 30 min after the beginning of the superfusion with l-[ 3H]tryptophan. A marked increase was seen in the serotonin release either in the presence of fluoxetine, a potent reuptake blocker of serotonin, or during depolarization with potassium chloride or veratridine. Calcium-free medium led to a decrease of spontaneous and potassium-evoked release. The veratridine-stimulating response was abolished by tetrodotoxin. Paradoxically, a slight increase in the spontaneous release of serotonin was observed in the presence of tetrodotoxin. Serotonin released from serotonin-containing SIF cells could be involved in the modulation of ganglionic transmission.