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).     

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 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.     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrine positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexus-like arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

Intrinsic innervation of the ewe cervix and its variations during pregnancy

Summary The course of the cholinergic and adrenergic nerve fibers in the cervix of the ewe was investigated in nonpregnant and pregnant animals using an acetylcholinesterase method and fluorescence histochemistry. Both technics in nonpregnant animals revealed a rich network of acetylcholinesterase and norepinephrin positive nerves around the blood vessels while the muscular innervation was moderately positive. Acetylcholinesterase fibers were also concentrated beneath the surface epithelium forming a plexuslike arrangement where isolated ganglion cells could be seen. At mid pregnancy cholinergic and adrenergic fibers decreased in density. The intensity of fluorescence was weaker and nerve fiber morphology was modified. We endeavoured to relate our findings to the problem of the neural control of contractions and the opening of the cervix of the ewe which is poorly supplied in nerve fibers, particularly at mid pregnancy.     

The autonomic innervation of the ovary of the dab,Limanda yokohamae

The autonomic innervation of the ovary of the dab was studied histologically and physiologically. The ovary receives a branch of nerve bundles that emerge into the abdominal cavity at the postero-ventral end of the kidney and can be traced back to the sympathetic chain in the vicinity of the 5th vertebra. Almost all the nerve fibers are AChE-positive, and some of them also emit adrenergic fluorescence. Electrical stimulation of the ovarian nerves caused ovarian contractions, and administration of ACh elicited contractions of the ovary preparations, supporting the hypothesis that the ovary is innervated by excitatory cholinergic fibers. In the ovarian nerve bundles, many AChE-positive and non-fluorescent ganglion cells are scattered. Ultrastructural studies suggest that nerve endings situated on the ovarian smooth muscle and on ganglion cells are cholinergic. These results also suggest that the cells are the post-ganglionic neurons of the cholinergic innervation and the axons of the cells reach to the muscle cells. On the other hand, the adrenergic fluoresecent fibers possibly participate in the inhibitory innervation, since the presence of inhibitory beta-adrenoceptors were demonstrated by pharmacological studies.     

Development of innervation to the atrial myocardium of the rabbit

Summary The development of innervation to the atrial myocardium of rabbits from 20th day of gestation to 35 days postnatal was studied ultrastructurally by electron microscopy and by demonstration of catecholamines by histofluorescence. Special attention was directed to the first morphologic appearance of nerve fibers and terminals and the closeness of juxtaposition of terminals with myocardial cells. Adrenergic and cholinergic terminals were identified on the basis of their differential ability to take-up and store the false adrenergic neurotransmitter 5-hydroxydopamine. Adrenergic terminals were first encountered at 20 days of gestation whereas cholinergic terminals could not be positively identified until the 24th day of gestation. Throughout development adrenergic terminals were more numerous than cholinergic, about 71 % of the terminals encountered being adrenergic. Many terminals approach closely (20–30 nm) to the sarcolemma of the muscle cells of the atrium. In many instances adrenergic and cholinergic fibers travel together in the same nerve bundle and are closely apposed without intervening Schwann-cell cytoplasm. Such a relationship could allow peripheral interaction between these fibers in the myocardium.Supported in part by the Kentucky Heart Association, Human Development Studies Program of the University of Kentucky and DHEW Grant 1 RO1 HL 22226-01 HED from the National Heart, Lung and Blood Institute. The technical assistance of Merle Wekstein is appreciated     

Innervation of the guinea pig spleen studied by electron microscopy

The innervation of the guinea pig spleen was investigated by electron microscopy. Unmyelinated nerve fibers in the capsulotrabecular and arterial systems were found to contain large and small granular and small agranular synaptic vesicles in their terminals and are thought to be sympathetic adrenergic in nature. They influence the contraction of the smooth muscle cells by diffusion innervation in these systems. These nerve terminals were also scattered in both the red and the white pulp. Pulp nerves wrapped by Schwann cells were further enclosed by myofibroblastic reticular cells. This condition revealed that the pulp nerves pass through the connective-tissue spaces of the reticular fibers, which contain elastic fibers, collagenous fibrils, and lamina densa-like materials of the usual basement laminae. One of the target cells for the pulp nerves is considered to be the myofibroblastic reticular cell in the reticular meshwork. Neurotransmitter substances released from the naked adrenergic nerve terminals travel through the reticular fibers and may play a role, by both close association innervation and diffusion innervation, in the contraction of reticular cells to expose the reticular fibers. At the exposed sides, connective-tissue elements of the reticular fibers are bathed with blood plasma, and the included naked nerve terminals, devoid of Schwann cells but with basement laminae of these cells, face free cells at some distance or are in close association with free cells, especially lymphocytes, macrophages, and plasma cells. The close ultrastructural relationship between the naked adrenergic nerve terminals and immunocytes strongly suggests that there is an intimate relationship between the immune system and the sympathetic nervous system through both close association innervation and diffusion innervation. Thus splenic adrenergic nerves of the guinea pig may play a triple role in 1) contraction of smooth muscle cells to regulate blood flow in the organ, 2) induction of the exposure of reticular fibers by contraction of the reticular cells in order to form a close relationship of the nerve terminals with the immunocytes, and 3) subsequent neuroimmunomodulation of the immunocytes.     

Analysis of nerve supply pattern in thoracic duct in young and elderly men

BACKGROUND: Analysis of the innervation pattern of the thoracic duct in young and elderly human subjects has been performed. The subdivision of the vessels in cervical and lumbar region were taken in consideration. MeTHODS AND RESULTS: Immunostaining for general nerve fibers with a PGP 9.5 marker disclosed a diffuse innervation of the thoracic duct in young subjects, which was strongly reduced in elderly subjects. In young subjects, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) immunoreactive fibers, markers of noradrenergic postganglionic sympathetic fibers, were frequent; choline acetyltransferase (ChAT) immunoreactive fibers, marker of cholinergic parasympathetic nerve fibers, were also well represented. Therefore, the influence of sympathetic and parasympathetic nerve systems on the thoracic duct can be confirmed. The immunoreactivity of vasoactive intestinal peptide (VIP), a neuropeptide frequently present in cholinergic parasympathetic nerve fibers, was scarcely present. Dopamine-positive fibers were observed in few short nerve fibers. Substance P (SP)-positive fibers were widely distributed in the medial and intimal smooth muscle layers, suggesting their involvement as contractile modulating fibers and sensitive fibers. In elderly subjects, an evident reduction of all specific nerve fibers analyzed was detected, the ChAT-positive fibers being the most affected. CONCLUSIONS: The lymphatic vessel thoracic duct is able to regulate hydrodynamic lymph flow by intrinsic contraction of its smooth muscle layer. Therefore, analysis of the thoracic duct innervation pattern may be important in assessing the regulation of vessel contraction. These findings called attention to the reduction of lymphatic drainage functionality affecting fluid balance in the elderly.     

The sphincter of the efferent filament artery in teleost gills: II. Sympathetic innervation

In addition to the cholinergic innervation described in the sphincter of the efferent filament arteries (Bailly and Dunel-Erb, ′86), an aminergic component has been demonstrated by specific techniques. The Falck fluorescence technique reveals a network of nerve fibers displaying a green fluorescence characteristic of catecholamines. At the ultrastructural level two types of fibers are present, one with clear vesicles and another with densecored vesicles. Axo-axonal synaptic relationships exist between the two types. Results of 5- and 6-OHDA (hydroxydopamine) treatments confirm the presence of an aminergic component. These observations support the notion of a dual innervation: cholinergic and adrenergic of, respectively, parasympathetic and sympathetic origin. The presence of presynaptic modulation is suggested. The aminergic component could inhibit or reduce the release of acetylcholine from cholinergic nerve endings. These results suggest that the sympathetic innervation modulates the vasoconstriction effect of the parasympathetic component.     

Histochemical studies of noradrenergic innervation of the liver in untreated and daunomycin-pretreated guinea-pigs

Summary The pattern of noradrenergic innervation of guinea-pig hepatic tissue was examined by using the fluorescence method of Falk et al. (1962). The distribution of monoaminergic nerve fibres in untreated animals was clearly evident at the level of the portal vein, the hepatic artery and the bile duct. The hepatic artery was the most densely innervated. Mast cells and Kupffer cells had no connection with the adrenergic fibres. In daunomycin-pretreated animals, the hepatic cells appeared to be deep red in colour owing to the presence of the antibiotic. Some noradrenergic nervous fibres, never previously described, were unexpectedly distinguishable in the liver parenchyma against the background red colour of daunomycin-pretreated tissue. They appeared to be contiguous to the hepatocytes and were of different shapes: comma-like, V-like, or like a string of pearls. The nature and the functional role of these elements is briefly discussed.     

Adrenergic terminal structures in the mesentery of mammals

This work is devoted to the study of adrenergic terminal structures in the mesentery of mammals (cat, dog). The investigation was performed with the Falck-Hillarp method of catecholamine fluorescence microscopy on total stretch mesentery preparations. The investigation showed that richly developed perivascular plexus constitute the basis of the adrenergic innervation system of the mesentery. In numerous points of these plexuses, single adrenergic fibers or polyaxonal structures are observed to issue into nonvascular areas of the mesentery where after repeated dichotomic division they pass into the preterminal and terminal parts. Being constructed on the principle of extended or restrained arborizations, these innervating structures have a morphological similarity with free sensory nerve endings. In this connection, the question of the possible existence of the sensory (afferent) links in the catecholamine-containing vegetative nerve plexuses is discussed.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Autonomic innervation of the ocular choroid membrane in the chicken

Summary The distribution pattern of adrenergic fibres innervating the ocular choroid membrane of the chicken was studied by means of fluorescence and electron microscopy. In addition, the origin of these fibres was investigated after superior cervical ganglionectomy. Adrenergic axons reach the choroid, partly forming the perivascular plexuses and partly running in the choroid nerves and the choroidal branches of the ciliary nerves. The axon terminals distribute to the smooth muscle cells of the arterial wall and to the extensive system of smooth muscle cells of the intervascular stroma. After unilateral ganglionectomy, fluorescent fibres almost completely disappeared, and degenerative changes could be observed in the terminal varicosities on both smooth muscle cell populations. These findings suggest that the adrenergic axons either originate from neurones within the ipsilateral superior cervical ganglion, or pass through this ganglion. The persistence of normal terminals in short- and long-term ganglionectomised animals shows that the vasal and intervascular muscle cells of the choroid membrane are provided with both an adrenergic and a cholinergic innervation.This work was supported by grant No 80.00442.04 from the Italian National Research Council (CNR)     

Adrenergic nerve fibers in the human fetal sciatic nerve.

The adrenergic innervation was studied in the human sciatic nerve at the gestational age of 16, 17, 18 and 21 weeks. Formaldehyde-induced catecholamine fluorescence, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) peroxidase-antiperoxidase immunohistochemistry methods were used. At the gestational age of 16, 17 and 18 weeks no adrenergic or NPY-positive nerve fibers were seen. At 21 weeks both fluorescence microscopy and TH immunohistochemistry showed adrenergic nerve fibers around arterioles in the epiperineurium and single nerve fibers in the endoneurium not related to blood vessels. The number of adrenergic nerve fibers appeared to be higher in the sciatic than in the tibial segment of the nerve. At this age, as at earlier stages of gestation, no NPY-positive nerve fibers were seen either in the epiperineurium or in the endoneurium. The results suggest that adrenergic nerve fibers may be associated with the epiperineurial blood vessels in the human sciatic nerve, and that the innervation starts to develop between 18 and 21 weeks of gestational age.     

Electron microscopic studies of the innervation of the human spleen

Summary The innervation of four normal human spleens was investigated by electron microscopy. Unmyelinated nerve fibers accompanied the arterial vascular system up to the arterioles of the red pulp. Neither myelinated nerve fibers nor ganglion cells were seen in the splenic hilum or in the splenic tissue itself. The nerve fibers terminated against the smooth muscle cells of the blood vessels in a manner that is typical of the autonomic nervous system. The terminal axons contained small and large granular vesicles and thus were adrenergic nerve fibers. In contrast to the results of previous studies using silver impregnation methods innervation of the red or white pulp could not be demonstrated. The findings on human spleens agree with those on mammalian spleens obtained by other authors using ultrastructural and fluorescence histochemical methods.We are indebted to Prof. Dr. K. Unsicker for his help in discussing the results     

The autonomic innervation of rat jugular vein

Summary The autonomic innervation of rat jugular vein was studied using glyoxylic acid fluorescence and acetylcholinesterase histochemical methods. The rat jugular vein is provided with both adrenergic and cholinergic nerve fibers organized in plexuses located at the adventitial-medial border. The existence of these nerve plexuses does not seem to support biochemical findings that suggest a lack of innervation in the rat jugular vein and which propose this blood vessel as a model for the analysis of drug-smooth muscle cell interaction without the interference of neuronal uptake mechanisms.     

Development of the innervation of fetal mesenteric microvasculature

Summary The innervation of the mesenteric microvasculature was studied in fetal and neonatal rabbits with the aid of methods demonstrating fluorescence of catecholamines and cholinesterase activity as well as a silver impregnation procedure. The results showed that: (1) adrenergic nerve fibers were present, coursing independently in the mesentery by day twenty-one of gestation, and were found routinely in the adventitia of arterioles and venules by day 25 of gestation; (2) cholinesterase positive cells and fibers of the myenteric plexus were present by day 18 of gestation but cholinergic fibers were not present in the mesentery until day 26; the latter not being associated with blood vessels; and (3) nerve fibers in the mesentery thought to be sensory stained positively with the Holmes silver method on day 18 of gestation.Supported by grants from the Akron Heart Association and the Heart Association of Southwestern Ohio.Recipient of N.I.H. Research Career Development Award AM-42, 370.     

Autonomic innervation of salivary glands in the armadillo,anteater, and sloth (Edentata)

The intraglandular distribution of adrenergic and cholinergic nerve fibers was studied histochemically in the parotid, mandibular, and sublingual glands of six species of edentates belonging to the three families that comprise the order; namely, the Dasypodidae (armadillos), the Myrmecophagidae (anteaters), and the Bradipodidae (sloths). The following histochemical techniques were used: (a) acetylcholinesterase reaction for the demonstration of cholinergic fibers; (b) formaldehyde- and glyoxylic acid-induced fluorescence for the demonstration of adrenergic fibers. In addition, norepinephrine (NE) was assayed fluorimetrically in the mandibular and parotid glands of the armadillo. A network of acetylcholinesterase-positive nerve fibers surrounds the intra- and interlobular ducts and endpieces of all glands; it is of low density in the mandibular and sublingual gland of the sloth, of high density in the sublingual gland of the anteater and of moderate density in the remaining glands. A vascular cholinergic innervation occurs in all salivary glands. Although present around the vessels, adrenergic new fibers were virtually absent from the parenchyma of all glands, even after in vitro incubation of glandular tissue with NE, or after administration of NE to armadillos previously treated with a monoamine oxidase (MAO) inhibitor. Consistent with this fact, the amount of NE present in the parotid and mandibular gland of the armadillo was extremely low. These findings may indicate that the salivary secretion in the edentates is regulated by the parasympathetic rather than by the sympathetic nervous system.     

Development of adrenergic neurons in uiuo: inhibition by ganglionic blockade

T he N ormal biochemical maturation of postsynaptic adrenergic neurons in mouse and rat superior cervical ganglion depends upon an intact preganglionic innervation (B lack , H endry and I versen , 1971a, 1972; T hoenen , S aner and K eitler , 1972). In recent studies tyrosine hydroxylase, the rate-limiting enzyme in norepinephrine biosynthesis (L evitt , S pector , S joerdsma and U denfriend , 1965), with localization to adrenergic neurons in the ganglion (B lack , H endry and I versen , 1971b), was used to monitor maturation of these cells. The developmental increase in tyrosine hydroxylase activity occurred simultaneously with the appearance of ganglionic synapses and was prevented by transection of the preganglionic nerve trunk (B lack , H endry and I versen , 1971a). These observations suggest that presynaptic cholinergic nerve terminals regulate the biochemical development of postsynaptic neurons in the superior cervical ganglion. The mechanism(s) by which presynaptic cholinergic terminals regulate postsynaptic development has not been elucidated. Such trans-synaptic regulation may be dependent on normal impulse transmission and/or may involve other unidentified, trophic factors. The results presented in the present communication suggest that normal development of ganglionic tyrosine hydroxylase activity is dependent on depolarization of postsynaptic adrenergic neurons.