Monoamine storage sites in the rat superior cervical ganglion following synthesis inhibition

Summary Monoamine storage sites in paraganglionic (PG-)cells of the rat superior cervical ganglion were investigated by electron and fluorescence microscopy following treatment with p-chlorophenylalanine (pCPA), disulfiram or guanethidine respectively.Dense core vesicles in PG-cells are significantly decreased (p< 0.001) in number following pCPA, and in the majority of these cells following disulfiram and guanethidine. However in a minor portion of PG-cells the latter agents cause an increase in number and in size of dense core vesicles, in parallel with structural alterations. In agreement with these electron microscopic findings fluorescence microscopic and cytophotometric evaluations reveal a general decrease in catecholamine content with few cells showing an increase.The findings provide a morphological basis for the assumption, that monoamine storage sites in PG-cells can be decreased by inhibition of monoamine synthesis, following administration of pCPA, disulfiram and guanethidine. However the two types of responses of PG-cells which occur after disulfiram and guanethidine demonstrate a functional heterogeneity of this cell system in the rat superior cervical ganglion which is discussed.Supported by Deutsche Forschungsgemeinschaft — Grant He 919/1.I like to thank Prof. Arnold, Tübingen, for the kind disposal of cytophotometric equipment.     

Influence of superior cervical ganglion stimulation frequency on salivary secretion in the rabbit. Comparative study of parotid and mandibular glands

Saliva secretion in response to the stimulation of the superior cervical ganglion (S.C.G.) at different frequencies (2, 3, 5, 10, 15, 20 Hz) has been studied in anaesthetized rabbits. The differences between the two major glands in this species were analyzed, with respect to the flow response, potassium, amylase and total protein content during the sympathetic stimulation. The stimulation of S.C.G. increased the salivary flow rate at all frequencies, on both parotid and mandibular gland. In the parotid gland the flow and stimulation frequency show a positive linear correlation which does not appear in the mandibular gland. In conclusion, the differences observed in the response to sympathetic stimulation in both glands seem to be due to distinct patterns of sympathetic innervation on different glandular elements.     

Granule-containing cells in the human superior cervical ganglion.

The fine structure of granule-containing cells in the human superior cervical ganglion is described. These cells are larger than the typical SIF cells in mammals and exhibit green-yellow fluorescence. They are characterized by numerous granular vesicles (80-140 nm in diameter) in the cytoplasm, but have many features in common with ordinary ganglion cells. They emit several long processes which form bundles together with ordinary nerve fibers. No synapses are found where the cells are presynaptic, although a few synapses are observed there where nerves are prosynaptic on the perikarya and processes of the cells. No close topographical relations are seen between the cells and blood vessels. It is suggested that the granule-containing cells are a special type of postganglionic aminergic neurons.     

Microvascularization of the rat superior cervical ganglion. A three-dimensional observation.

The three-dimensional image of the microvascularization of the rat superior cervical ganglion (SCG) was examined using the vascular corrosion cast technique in conjunction with scanning electron microscopy. It was found that the rat SCG was a highly vascularized organ. Arteries supplying the ganglion gave rise to a subcapsular capillary plexus before branching off to become intraganglionic capillaries. Two types of intraganglionic capillaries, large and small, were observed throughout the organ. Numerous anastomoses among these capillaries were found before they converged into venules and collecting veins. However, a pattern of blood vessels resembling portal-like intraganglionic microcirculation could not be demonstrated.     

Folate metabolism in the superior cervical ganglion histochemical study.

By means of histochemical methods, folic acid, dihydrofolate reductase and NADH2-cytochrome-C-reductase were studied in the bovine superior cervical ganglion, in parallel with quantitative estimations of dihydrofolate reductase activity and in connection with the process of ageing. Various levels of folate metabolism were present in nerve cells and glial cells, as well as in pre or postganglionic nerves. In the process of ageing the activity of dihydrofolate reductase gradually decreased and the folic acid concentration in the nerve cells increased. Thus the enzyme --- substrate ratio appeared to favour the enzyme in young animals but the substrate in old animals.     

Origins of the afferent fibers to the cat superior cervical ganglion.

The origins of the afferent fibers to the cat's superior cervical ganglion (SCG) were demonstrated by using the retrograde horseradish peroxidase tracing method. We found that the preganglionic neurons were located in the spinal segments C8-T5, particularly in T1-T3. These neurons were situated mainly in the intermediolateral column. The extra-SCG neurons along with the cervical sympathetic trunk originated ipsilaterally from the middle cervical and stellate ganglia, and contralaterally from the caudal part of the SCG. Labeled neurons also originated from the mandibular division of the trigeminal ganglion. Our results demonstrated that many fiber sources projected to the SCG, which plays a complicated synaptic role in controlling the visceral organs of the head and neck region.     

Electron microscope localization of acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat. I. Normal ganglion

The distributions of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the superior cervical ganglion (SCG) of the cat were determined by electron microscopy (EM) with the bis- (thioacetoxy)aurate (I), or Au(TA)2, method. Before the infusion of fixative, one of the enzymes was selectively, irreversibly inactivated in vivo, as confirmed by light microscope (LM) examination of sections of the stellate ganglion stained by the more specific copper thiocholine method. Physostigmine-treated controls, for inhibition of AChE or BuChE, were stained concomitantly with tissue for enzyme localization by the Au(TA)2 method for EM examination in each experiment. It was concluded that most of the AChE of the cat SCG is present in the plasma membranes of the preganglionic axons and their terminals, and in the dendritic and perikaryonal plasma membranes of the postsynaptic ganglion cells. BuChE is confined largely to the postsynaptic neuronal plasma membranes. Reasons for the discrepancies between the localizations found by the present direct EM observations and those deduced earlier from LM comparisons of normal and denervated SCG are discussed. It is proposed that a trophic factor released by the preganglionic terminals is probably required for the synthesis of postsynaptic neuronal AChE, and that BuChE may serve as a precursor of AChE at that site.     

Acetylcholine content in superior cervical ganglion following reinnervation of vagal afferent fibers in cat

The superior cervical ganglion (SCG) was reinnervated by vagal afferent fibers by cross anastomosis between the cranial end of nodose ganglion and the caudal end of SCG in cats. Formation of functional synapses was evidenced by unilateral mydriasis and contraction of the nictitating membrane in response to inflation of the stomach with a balloon or to electrical stimulation of the afferent vagus. The acetylcholine (ACh) content in the cross-anastomosed SCG (reinnervated by vagal afferent fibers) was measured. In anastomosed SCG, the ACh content was about half of normal SCG, but significantly higher than chronically decentralized SCG. Also the ACh content in nodose ganglion (NDG) was investigated in situations in which there was anastomosis, chronic supra, infra, or supra-/infranodose vagotomy. The ACh content of anastomosed NDG was near that of supranosdose vagotomized ganglion. The ACh content of supra-/infranodose vagotomized NDG, which can be considered the NDG itself, was as much as that of normal intact NDG. It was found that the ACh content of infranodose vagotomized NDG was increased, possibly the result of vagal efferent axonal flow or transport. The ACh content of vagal trunk with or without infranodose vagotomy was also measured. The ACh content of vagal trunk with infranodose vagotomy was smaller than that of the normal trunk, but there was still a considerable quantity of ACh. There was no significant change in wet weight of the SCG and NDG before or after the operations. From these results we have concluded that the transmission of the cross-anastomosed SCG (reinnervated with vagal afferent nerve) was cholinergic; and that the vagal afferent nerve have afferent cell bodies not only in NDG but also in peripheral vagal trunks (infranodose portion). These results strongly suggest that vagal afferent fibers are in part cholinergic.     

Electron microscope localization of acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat. II. Preganglionically denervated ganglion

Cat superior cervical ganglia (SCG), denervated preganglionically 6-8 d previously, were stained for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by the bis-(thioacetoxy)aurate (I), or Au(TA)2, method and compared by electron microscopy with normal SCG described previously (Davis, R., and G. B. Koelle. 1978. J. Cell Biol. 78:785-809). In confirmation of earlier light microscopic findings by the highly specific copper thiocholine method, there was nearly a total disappearance of AChE from the ganglion; no myelinated or unmyelinated axons with AChE-stained axolemmas were found, and only occasional traces of AChE staining were noted at dendritic and perikaryonal plasma membranes. Considerable staining for BuChE persisted at the latter sites, however. As in the normal SCG, physostigmine-resistant staining, caused by noncholinesterase enzymes plus the possible presence of very low concentrations of AChE or BuChE, was noted at external mitochondrial membranes, elements of the endoplasmic reticulum of neurites and Schwann cells, and also in lysosomes. These findings confirm the previous identification of AChE-stained myelinated fibers in the normal SCG as preganglionic and of the unstained myelinated fibers as postganglionic. It is proposed that the maintenance of AChE at postsynaptic sites in normal ganglia is caused by the release of a trophic factor(s) from presynaptic terminals. The source of the postsynaptic BuChE, which is apparently completely absent from the endoplasmic reticulum of the ganglion cells, remains unexplained.     

Transmission pathways in the rat superior cervical ganglion

On isolated preparations of the superior cervical ganglion (SCG, n = 8) taken from 21-day-old rats, we studied the intraganglion pathways and mechanisms underlying generation of synaptic responses of SCG neurons to antidromic stimulation. One of the three nerves connected with the SCG was stimulated, and compound action potentials were recorded simultaneously from the other two nerves; then, the order of stimulated and recorded nerves was changed. Orthodromic stimulation of the cervical sympathetic nerve (CSN) evoked responses in the internal carotid nerve (ICN), which could be completely blocked by hexamethonium, and responses in the external carotid nerve (ECN), which contained a component that was not blocked by this of the ECN caused responses in the CSN, which were not blocked by hexamethonium. Effects of superfusion of the SCG with a Ca²⁺-free solution allowed us to conclude that the hexamethonium-insensitive component of the responses in the CSN and ECN and ECN-CSN conduction can be explained by the presence of direct fibers going from the CSN to the ECN with no synaptic relay. Possible mechanisms underlying antidromic stimulation-induced synaptic responses in SCG neurons are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 396–399, July–October, 2007.     

Effects of ganglion blocking agents on post-train facilitation in the rabbit superior cervical ganglion

The effect of ganglion blocking agents, hexamethonium and tubocurarine, on post-train facilitation and ganglionic transmission was studied and compared in isolated superior cervical ganglion of the rabbit, using electrophysiological technique--the conditioning-testing methodology. The preganglionic nerve trunk was stimulated, with either a single unconditioned stimulus (UR)-or a train of conditioning stimuli at 10 or 30 Hz, followed by a post-train test stimulus (PTR). The transmitted postganglionic, compound action potential (PCAP) was recorded following single and trains of stimuli, in the presence and absence of ganglion blocking drugs, hexamethonium (1-100 microM) and tubocurarine (1-100 microM). Hexamethonium and tubocurarine produced concentration-dependent reduction in the amplitude of the transmitted PCAP, increased post-train facilitation values and proportionately reduced those of the subliminal fringe (SF). The mean IC50 values (concentration to produce 50% block of PCAP) of hexamethonium and tubocurarine-induced blockade of the single unconditioned response were 15 +/- 1 microM and 26 +/- 2 microM (n = 6, P less than 0.01) respectively. A dose-ratio (tubocurarine)/hexamethonium) of 1.7 was obtained.     

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [³²P]P_i and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. ³²P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of ³²P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of ³²P into tyrosine hydroxylase, with 50 μM veratridine producing a 5-fold increase in ³²P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 μM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca²⁺. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of ³²P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.