Electron microscopic investigation of adrenergic and non-adrenergic axons in the rabbit SA-node

Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO₄ which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.This work was supported by grants from Åhlén-Stiftelsen, the Faculty of Medicine, University of Lund, Lund, Sweden, the United States Public Health Service (project 06701-04) and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05).     

Recent studies of the actions of cholinomimetic drugs on adrenergic nerves and their implications for the cholinergic link hypothesis

This review analyzes the results of recent studies of the actions of cholinomimetic drugs on adrenergic nerve terminals and their implications for the cholinergic link hypothesis. Thus far, evidence suggests that the only possible action of endogenous acetylcholine (ACh) present near noradrenaline (NA) stores is an inhibition of the release of NA from the adrenergic nerve terminals and that NA is released only when the action of acetylcholinesterase is inhibited. Nicotinic agents have been shown to act on adrenergic nerve terminal membranes, a finding that casts doubt on the proposed intraneuronal cholinergic sites for the action of endogenous ACh. Evidence also indicates that the mode of adrenergic neurone blocking action of bretylium and guanethidine is independent of the proposed cholinergic process in NA release. Current findings do not support the proposal that nicotinic agents in higher concentrations interfere with adrenergic neurotransmission. It is therefore concluded that nicotinic agents, in causing the release of NA from adrenergic nerve terminals, are merely exhibiting a pharmacological action and not mimicking the physiological function of ACh, as proposed by the cholinergic link hypothesis.     

Studies on the acetylcholinesterase (ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA)treated rat iris

Summary The adrenergic and cholinergic innervation to the rat iris has been studied at a light and electron microscopic level. Catecholamine fluorescence histochemistry showed adrenergic nerves to be present in both the dilatator and the constrictor pupillae regions. At a fine structural level the terminal innervation of the iris was studied and criteria for the differentiation between presumptive adrenergic and presumptive cholinergic axon terminals were examined. To aid this examination presumptive adrenergic axons were either labelled with the false adrenergic transmitter, 5-hydroxydopamine, or chemical sympathectomy performed using 6-hydroxydopamine. The value of using acetylcholinesterase staining as a marker for cholinergic nerve terminals was also studied.Results showed a mixed adrenergic/cholinergic innervation to the dilatator pupillae. In the constrictor pupillae an exclusively cholinergic innervation was found although adrenergic and cholinergic nerves were found supplying the blood vessels and at the dilatator-constrictor interface. These findings are discussed with regard to innervation-function relationships in the iris.     

Cholinergic and adrenergic innervation of the bladder obstructed by urethral stenosis in the rat

In the obstructed bladder by urethral stenosis the morphometry studies show a decrease of the cholinergic and adrenergic intrinsic innervation in a different way according the tissue layer and the experimental delay. Adrenergic nerves are more affected than cholinergic ones but they recover more quickly. In the adventitia and the sub-mucosa first affected by the high pressures inside the bladder the density of the autonomous nerves decreases from the first week. The perivascular and muscular nervous fibers are less sensitive and the similar shape of their innervation histogram gives evidence of a tight bond between their adrenergic network. Beside their motor participation the adrenergic terminal fibers may play a role in the cellular trophicity of the muscle.     

Ultrastructure of the basiepithelial nerve plexus of the sea urchin,Centrostephanus longispinus

Summary In submandibular glands of rabbits both adrenergic and cholinergic axons are intimately associated with parenchymal cells of the intercalary ducts and the granular tubules, lying beneath the basement membrane and often in the space between the parenchymal cell and an associated myoepithelial cell. The submandibular acini receive a less intimate and less plentiful innervation by adrenergic and cholinergic axons which remain outside the basement membrane and are still associated with Schwann cells. Occasional axons of both adrenergic and cholinergic type occur beneath the basement membrane of submandibular striated ducts in intimate association with basal parts of the cells.In the parotid glands numerous adrenergic and cholinergic axons are found beneath the basement membrane of acini and intercalary ducts in intimate association with the cells.This work has been helped by the technical assistance of Mr. P.S.A. Rowley     

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

Summary The relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.This work was supported by grants from the United States Public Health Service (06701-04), the Faculty of Medicine, University of Lund, Lund, Sweden and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05). Svenska sällskapet för medicinsk forskning.     

Ultrastructure of cholinergic innervation in the cirrhotic liver in guinea pigs. Neurohistochemical and ultrastructural study

Hepatic cirrhosis was induced in guinea pigs by ligation of the common bile duct and innervation of the liver was studied by fluorescence histochemistry (glyoxylic acid method), acetylcholinesterase (AChE) neurohistochemistry (modified Karnovsky and Roots method), and transmission electron microscopy. In control animals the adrenergic terminals showed connections with endothelial cells, hepatocytes and fat-storing cells, but no cholinergic terminals were evident. Cirrhosis was present 6 weeks after the bile duct ligation and marked fibrosis, accompanied by bile duct proliferation, was evident in the portal areas. Numerous AChE-positive nerve fibers traversed the collagenous bundles in the fibrotic areas, and cholinergic terminals formed close contacts with fibroblasts. Each axon terminal was found to contain numerous small coreless vesicles and AChE-reaction products were confirmed in the space between a nerve terminal and a fibroblast. In contrast, fluorescence adrenergic nerve fibers and their terminals remained unchanged. This study demonstrates that parasympathetic cholinergic innervation participates in some stages in the development of hepatic cirrhosis.     

Pharmacological reactions of the circular muscle of the guinea pig vas deferens.

Pharmacological responses of spiral strips prepared from the guinea pig vas deferens to various adrenergic and cholinergic agonists and autacoids were studied. On the circular muscle alpha adrenergic, muscarinic cholinergic and histaminergic receptors were identified. The responses evoked on the circular and longitudinal muscles were of the same type.     

Innervation of iridic melanophores

Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.This work has been supported by grants from Lunds Läkarsällskap, the Swedish Medical Research Council (Project no. B69-14X-2321-02 and B69-14X-712-04C) and NIH (06701-02).     

The major ganglion in the pelvic plexus of the male rat

Summary To further evaluate the role of autonomic ganglia in the regulation of pelvic visceral activity, the neural elements in the major pelvic ganglion of the male rat have been studied with histochemical and electron microscopic techniques. The principal findings are that the ganglion is composed of cholinergic and adrenergic ganglion cells as well as small intensely fluorescent (SIF) cells. Polarity in the ganglion is indicated by clustering of small ganglion cells which stain intensely for acetylcholinesterase (AChE) along the pelvic nerve while larger cells, with weak to moderate AChE activity, collect near small branches of the hypogastric nerve. Some cholinergic ganglion cells are enclosed by a plexus of adrenergic terminals. SIF cells appear to be in contact with both cholinergic and adrenergic cells, although many of the fluorescent beads around adrenergic neurons may be short dendrites of ganglion cells, rather than processes of SIF cells. Two types of SIF cells may be distinguished on the basis of size and morphology of their granulated vesicles. Afferent synapses of the cholinergic type were common on SIF cells of the large granule and small granule type. Portions of SIF cells with large granules occur within the capsule of ganglion cells. Contacts seen here were interpreted as efferent synapses from SIF cells to the dendrites of ganglion cells.     

The development of innervation in the rat atrioventricular node

Summary The problem of development of the innervation of the rat atrioventricular node has been investigated by electron microscopy. Nerve bundles appear in relation to the node as early as the second postnatal day and vesiculated axons are seen throughout the entire node by the fourth day. Intimate contacts between nodal cells, axons and terminal varicosities are frequently observed.Use of the 5-hydroxydopamine tracer technique has enabled the identification of both cholinergic and adrenergic axons. It is concluded that the node has a dual innervation although cholinergic endings far outnumber those classified as adrenergic on the sixth postnatal day.These results are quite different to earlier findings made at the light microscope level and the discrepancies are discussed with respect to the histochemical techniques used. The suggestion that nodal differentiation is induced by nerves is considered in relation to the differences in cholinesterase activity exhibited by nodal cells during normal development and following neonatal sympathectomy.     

Adrenergic and cholinergic innervation of the prostate in laboratory animals

The prostate innervation has been studied in 50 white rats, 12 rabbits, 12 guinea pigs, 6 cats and 6 dogs. Together with the impregnation techniques, Karnovsky-Roots method has been applied, for revealing cholinergic components, and the incubation method in 2% solution of glyoxylic acid, for revealing adrenergic nervous structures. Density of adrenergic and cholinergic nervous plexuses has been estimated by means of the planimetric point method. The prostate of the laboratory animals possesses well manifested adrenergic and cholinergic nervous plexuses. The organ's alveolus and ducts are covered with adrenergic and cholinergic fibers, however, the relative density of the cholinergic plexuses is less than that of the adrenergic ones. The guinea pig prostate is the most richly supplied with the adrenergic nervous plexuses, and the rabbit prostate--with the cholinergic nervous plexuses.     

Catecholamine-containing nerve terminals of the eccrine sweat glands of macaques

Summary A loose network of catecholamine-containing nerves was demonstrated with a fluorescence histochemical method (Falck-Hillarp) in the coiled portion of eccrine sweat glands in the digital pads of macaques after the injection of nialamide and noradrenaline. In the skin of untreated control animals, fluorescent fibers appear only in some of the glands. A systemic administration of reserpine and a local injection of 6-hydroxydopamine (6-OHDA) or 5-hydroxydopamine (5-OHDA) into the digital pad cause a complete disappearance of fluorescent fibers around the glands and blood vessels. Electron micrographs reveal many unmyelinated varicose axon profiles outside the basement membrane of secretory tubules. Most of these profiles contain many small agranular vesicles and a few large dense-cored vesicles (cholinergic terminal), and some have numerous small granular and a few large densecored vesicles (adrenergic terminal).The local injection of 6-OHDA causes various degenerative changes in the adrenergic terminals but the cholinergic ones and the rest of the cellular structure remain intact. The injection of 5-OHDA induces a significant increase of electron-dense granules in the vesicles of adrenergic terminals.The presence of catecholamine and the effects of 6-OHDA and 5-OHDA in the nerve terminals indicate that the innervation of the eccrine sweat glands of macaques consists of cholinergic as well as adrenergic terminals.Publication No. 783 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grant RR 00163 of the Animal Resources Branch, Division of Research Resources.We acknowledge the excellent assistance of Tsutomu Yoshida, Tsuneka zu Fuse, John Ochsner, and Nickolas Roman.     

Studies of cardiac ganglia in pre- and postnatal rabbits

Summary This investigation was undertaken to describe the ultrastructure of cardiac ganglia in rabbits from day 18 of gestation to day 35 postpartum. Special attention was directed to the types of synaptic contacts made with the principal neurons and with the small granule-containing cells. The cardiac ganglia in all animals consisted mainly of parasympathetic postganglionic neurons, supporting cells, and small granule-containing (small intensely fluorescent) cells. The neurons received afferent synaptic terminals of two types. One type contained mainly small clear vesicles typical of most cholinergic terminals. The second type contained mainly small dense-core vesicles (these were most prominent after treatment of the animal with 5-hydroxydopamine), and were considered to be adrenergic terminals. These adrenergic terminals are probably part of an inhibitory system in the ganglia. The small granule-containing cells received typical afferent synaptic terminals of the cholinergic type, and also formed specialized contacts with certain axonal terminals. These latter specializations are considered to be reciprocal synapses which probably have a role in modulating ganglionic transmission.Supported by the Kentucky Heart Association and the Heart Association of Louisville and Jefferson County     

Complexities and sequence similarities of mRNA populations of cholinergic (NS20-Y) and adrenergic (N1E-115) murine neuroblastoma cell lines

The clonal murine neuroblastoma cell lines NS20-Y and N1E-115 have been proposed as models for examining the commitment of neural crest cells to either the cholinergic or adrenergic phenotype, respectively. The validity of this model depends in part on the extent to which these two cell lines have diverged as a result of their transformed, rather than neuronal properties. In order to quantitate differences in gene expression between NS20-Y and N1E-115 cells, the mRNA complexity of each cell type was determined. An analysis of the kinetics of hybridization of NS20-Y cell mRNA with cDNA prepared from NS20-Y cell mRNA demonstrated the presence of approximately 11,700 mRNA species assuming an average length of 1900 nucleotides. A similar analysis using mRNA isolated from N1E-115 cells and cDNA prepared from N1E-115 cell mRNA demonstrated that the adrenergic cell line expressed approximately 11,600 mRNA species. The species of mRNA expressed by each cell line were resolved into high, intermediate, and low abundance populations. In order to determine whether mRNAs were expressed by the cholinergic, but not by the adrenergic cell line, NS20-Y cDNA was hybridized to an excess of N1E-115 cell mRNA. An analysis of the solution hybridization kinetics from this procedure demonstrated that the two cell lines do not differ significantly in the nucleotide complexity of their mRNA populations. The extensive similarity between the two mRNA populations suggests that only a small number of genes are expressed differentially between the two cell lines and supports their use as models for the differentiation of cholinergic and adrenergic neurons.     

Parasympathetic and sympathetic postganglionic synapses in ureterovesical autonomic pathways

Summary Histochemical techniques for acetylcholinesterase and catecholamine show that ureterovesical ganglia of both cat and dog contain dense intraganglionic cholinergic and adrenergic plexuses. Ramifications of both plexuses surround most cholinergic and adrenergic ganglion cell bodies as pericellular synaptic plexuses. Similar pericellular plexuses exist around extraganglionic cholinergic and adrenergic ganglion cells. Both adrenergic and cholinergic synaptic fibers persist in denervated pregnaglionic nerve-free specimens, indicating that cholinergic synaptic fibers are postganglionic parasympathetic in nature. The presence of adrenergic (postganglionic sympathetic) and postganglionic parasympathetic synapses around cell bodies in ureterovesical ganglia provides a morphologic basis for the sympathoinhibitory and muscarinic parasympatho-excitatory phenomena described in these ganglia.     

Differential functional relevance of a plasma membrane ganglioside sialidase in cholinergic and adrenergic neuroblastoma cell lines.

Gangliosides located in the outer leaflet of the plasma membrane are important modulators of cellular functions. Our previous work has shown that in cultured human SK-N-MC neuroblastoma cells a sialidase residing in the same membrane selectively desialylates gangliosides with terminal sialic acid residues, causing a shift from higher species to GM1 and a conversion of GM3 to lactosylceramide. Inhibition of this sialidase by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en) resulted in increased cell proliferation and a loss of differentiation markers. In this study, we examined the occurrence and function of this ganglioside sialidase in other neuronal cells. Subcellular fractionation showed the sialidase to be located in the plasma membrane of all cell lines studied. The presence of the inhibitor NeuAc2en led to a profound decrease in the amount of the differentiation marker 200 kDa/70 kDa neurofilaments and an increase in cell proliferation in the cholinergic SK-N-MC and mixed cholinergic/adrenergic SK-N-FI and SK-N-DZ neuroblastoma lines, but had little or no effect in the human adrenergic SK-N-SH and SK-N-AS and the adrenergic/cholinergic PC12 cells from rat. The influence of the inhibitor on cell behaviour was paralleled by a diminished number of cholera toxin B-binding GM1 sites. The findings demonstrate that the plasma membrane ganglioside sialidase is an important element of proliferation and differentiation control in some, but not all, neuroblastoma cells and suggest that there might be a relationship between plasma membrane sialidase activity and cholinergic differentiation.     

Electron microscopic and histochemical study of the localization of adenylate cylase and acetylcholinesterase in the synapses of the rat cerebral cortex and basal ganglia]

Synapses of the brain cortex and basal ganglia of the rat were studied by means of electron histochemical reactions to adenylate cyclase and acetylcholinesterase. Three types of synapses, viz. cholinergic, adrenergic and mixed, were identified. Typification was carried out on the basis of presynaptic terminal vesicle characteristics and localization of the mentioned enzymes of the synapse receptor area.     

Development of nerves plexuses in the pelvis of the prenatal rabbit

By means of neurohistological and histochemical methods using serial sections of the embryos at the age of 12-30 days of development, the premediatory and beginning of the mediatory stages in development of cholinergic and adrenergic neural plexuses have been studied in rabbit small pelvis. Appearance of catecholamine-containing cells at early mediatory stages and a gradual development and complication of the cholinergic and adrenergic neural plexuses structure are demonstrated. By birth in the rabbit small pelvis area, some elements of cholinergic and adrenergic structures begin to form.     

The innervation of the uterus in Salamandra salamandra (L.) (Amphibia,Urodela)

The innervation of the uterus in the ovoviviparous urodele Salamandra salamandra was studied. In whole mount preparations of the thin-walled uterus of pregnant females, a dense adrenergic network was demonstrated using a modified glyoxylic acid fluorescence technique. Based on vesicle type and cytochemical reactivity after chromate/dichromate fixation for electron microscopy at least two types of neural process were distinguished and classified as adrenergic and cholinergic. Both types are preferentially situated above or between the smooth muscles of the uterine tissue. Adjacent to the muscles in the walls of arterioles mainly adrenergic fibers are seen. Using high performance liquid chromatography with electrochemical detection in tissue homogenates of uterus a considerable amount of noradrenaline could be identified. The significance of the dense innervation is discussed with respect to the function of the uterus during pregnancy and birth.