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).     

Intraovarian adrenergic nerves in the guinea-pig: Development from fetal life to sexual maturity

Summary The development of the intraovarian adrenergic nervous system was investigated in the guinea-pig by use of chemical determination of catecholamines with high performance liquid chromatography (HPLC) and with the formaldehyde-induced fluorescence method for visualization of adrenergic nerves (Falck-Hillarp technique). Ovaries from fetuses (39–40, 45–50, 55–57, 60–63 days of gestation) and young animals (1, 2, 3, 7, 14, 30, 40–45 days of age) were included in the study. The noradrenaline concentration was low in the ovaries from the youngest fetuses but increased with age, reaching a maximum level at 2 days post partum. A marked decrease in noradrenaline concentration from the second to the third day of life was found as a consequence of the rapid increase in the ovarian weight during this time. A similar decrease in ovarian noradrenaline concentration after a period of rapid ovarian growth was noted at 30 days of age. Measurable amounts of adrenaline were found in the ovary only in the fetal stages; the highest concentration (0.73 g) was detected at 55–57 days of gestation.     

Occurrence of adrenergic nerve fibers in human thymus during immune response

The adrenergic nerve fibers (ANF), the neuropeptide Y-like immunoreactive nerve fibers (NPY-NF) and the noradrenaline (NA) amount were studied in the human thymus in subjects previously treated or not treated with interferon therapy with the aim to identify the changes due to the interferon therapy. This therapy has been used in patients affected by multiple sclerosis (MS). Biochemical and morphological methods were used associated with quantitative analysis of images. The whole thymuses were removed during autopsies in young and adult patients not treated with interferon. Moreover, samples of thymus were removed from patients, either young or adult who had previously been treated with interferon therapy, and subjected, for diagnostic reasons, to thymic biopsy. All samples of thymus were weighed, measured and dissected. Thymic slices were stained with Eosin-orange for detection of the microanatomical details, or with Bodian's reaction for recognition of nervous structures. Histofluorescence microscopy was used for detection of ANF, and immunofluorescence microscopy for recognition of NPY-like immunoreactive structures. All morphological results were subjected to quantitative analysis of images. Noradrenaline contained in thymic structures was measured by biochemical methods. Our results only concerned the effects of the therapy and suggested that treatment with interferon therapy induces many changes in the thymic structures: (1) The protein content of thymus is significantly increased; (2) the NA content in the thymus is also significantly increased; (3) NPY-like immunoreactive structures in the thymus are significantly increased; (4) occurrence of NPY-like immunoreactivity is particularly and significantly increased both in thymic microenvironment and in structures resembling nerve fibers; (5) ANF are significantly increased in the same thymic structures in which NPY-like immunoreactivity is also increased (i.e. thymic microenvironment and structures resembling nerve fibers). The morphological and biochemical changes observed can also explain the immunological changes induced in the thymus after immunostimulating therapy.     

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.     

The origin and distribution of adrenergic nerve fibres in the guinea-pig colon

Summary A detailed study of the origin and distribution of sympathetic fibres in the distal colon of the guinea-pig has been made using the fluorescent histochemical method for localizing catecholamines. The extrinsic adrenergic fibres of the colonie sympathetic nerves follow the inferior mesenteric artery and its branches to the colon. Some of the extrinsic adrenergic fibres are associated with the parasympathetic fibres of the pelvic nerves near the colon. Complete adrenergic denervation follows the removal of the inferior mesenteric ganglion or the destruction of the nerves running with the inferior mesenteric artery.No fluorescent fibres, other than those associated with blood vessels, were observed in air-dried stretch preparations of the isolated longitudinal muscle. However, a substantial number of varicose, terminal fibres, not associated with blood vessels, were observed in the circular muscle. Some varicose fibres, apart from those associated with ganglion cells, were observed in the myenteric plexus. These fibres were seen in the bundles of nerves running between the nodes of the plexus and also as single fibres which branched from the plexus to end in areas free of ganglion cells.Three plexuses of adrenergic nerve fibres have been distinguished in the submucosa: a dense plexus of terminal fibres innervating both the veins and arteries; a plexus consisting of innervated nodes of ganglion cells, connected by bundles of fluorescent and non-fluorescent nerves; and a plexus of varicose and non-varicose fibres, which is not associated with ganglion cells. Some groups of ganglion cells in the submucosa were without adrenergic innervation.A plexus of varicose fibres forms a meshwork in the lamina propria of the mucosa. The muscularis mucosae is sparsely innervated. Most of the blood vessels in the mucosa are not associated with adrenergic fibres.     

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     

Development of the innervation in the human pineal organ

Summary This investigation is concerned with pineal organs of human embryos 60 to 150 days old. At every stage central nerve fibres enter the pineal organ by way of the habenular commissure, but are restricted to the pineal's proximal part. On about the 60th day of the development the sympathetic nervus conarii grows into the distal pole of the pineal organ from a dorso-caudal direction and plays the predominant part in the innervation of the pineal organ. After penetrating, it soon branches out and forms a network in the pineal tissue. Much later, not until the 5th embryonic month, sympathetic nerves appear accompanying the supplying vessels in the perivascular spaces. After a short time these nerves pierce the outer limiting basement membrane and penetrate the parenchyma. Towards the end of the 5th embryonic month the axons of the sympathetic nerves form varicosities containing clear and dense core vesicles. At this point large amounts of laminated granules appear primarily in cell processes, probably of pinealocytes. Isolated granules also occur in the varicosities of axons. The granules encountered here are most likely secretory granules.Dedicated to Professor Bargmann on his 65th birthday.     

The bovine tubouterine junction: general innervation pattern and distribution of adrenergic,cholinergic, and peptidergic nerve fibers

The innervation of the bovine tubouterine junction was studied in sexually mature heifers using antisera against various neuronal markers and a modified acetylcholinesterase method. The vast majority of the nerve fibres in the bovine tubouterine junction belongs to the sympathetic nervous system; peptidergic and cholinergic fibers are restricted to characteristic locations. The endosalpinx in the adovarian portion of the terminal tubal segment is poorly innervated. The mucosa of the aduterine portion and of the tubouterine transitinal region proper receives a strikingly dense innervation, which is observed mainly in combination with a strong vascularisation of specialised mucosal structures. In the endometrium, perivascular nerves accompany the ascending spiral arteries but sporadic contacts between nerve fibres and uterine glands are also observed. From the muscular coat the inner longitudinal layer of the terminal tubal segment is more richly supplied by nerve fibres than the intermediate circular and outer longitudinal layers of the tubouterine junction. No changes in the innervation pattern were seen during the different stages of the sexual cycle.     

A stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibres in the mouse peripheral tissues after chemical sympathectomy with 6-hydroxydopamine

Summary Systemically administered Nerve Growth Factor (NGF) had a strong stimulating effect on the regeneration of fully developed adrenergic neurons in the peripheral tissues of the mouse after axotomy induced by 6-hydroxydopamine. The NGF stimulation was investigated at 9 and 21 days after the 6-hydroxydopamine injection, and was observed fluorescence histochemically as an increase in number, length, and thickness of the outgrowing adrenergic fibre bundles, in the extent and abundance of the terminal ramifications of the regrowing fibres, and in their fluorescence intensity. This increase in the regrowth of the lesioned adrenergic axons was paralleled by strong and significant increases in the recovery of endogenous noradrenaline in several peripheral tissues. The present findings demonstrate a sensitivity of fully developed adrenergic neurons to NGF during axonal regeneration, and it is suggested that NGF might play a normal physiological role in this process.     

The effect of decentralisation or chronic hypogastric nerve stimulation in vivo on the innervation and responses of the guinea-pig vas deferens

Summary The physiological, pharmacological and morphological characteristics of guinea-pig vas deferens supplied by hypogastric nerves rendered inactive by decentralisation were compared with those of vas deferens in which the nerve supply had been chronically stimulated for 3–9 days using implanted electrodes. No change was seen in decentralised preparations prior to 7 days, but from 8–15 days, increased sensitivity to application of noradrenaline in vitro was observed, which was shown to be related to reduced transmitter uptake by nerve terminals as well as to an increase in postjunctional sensitivity; there was also increased fatigability 7–14 days following decentralisation. Continuous stimulation of hypogastric nerves at 2 Hz for 4–8 h daily for 4–8 days resulted in enhanced transmitter uptake and reduced responses to noradrenaline; this was associated with a slight increase in noradrenaline content and a faster adrenergic neuromuscular response with a shorter latency. No appreciable changes in nerve or muscle structure studied by electron microscopy were observed following decentralisation, but there was an increase of between 12.5 and 29.6% in the number of close (< 100 nm) neuromuscular junctions following chronic stimulation for 8 days.     

Adrenergic innervation of the tunica media in the saphenous artery of the fetal and newborn guinea-pig

Summary Previous studies have demonstrated that adrenergic nerves are located in the medial-adventitial border of the muscular arteries. Observations made in this study have revealed that adrenergic nerves penetrate into the outer medial layer of the saphenous artery in fetal and newborn guinea-pigs, while in the adult these nerves are located in the medial-adventitial border. It is proposed that the adrenergic nerves located in the tunica media may have a trophic effect on the medial smooth muscle. It is further suggested that the final refinement of the dual control system of arterial walls, by nerves and circulating catecholamines, involves exclusion of adrenergic nerves from the tunica media.     

Ultrastructural observations on the central innervation of the guinea-pig pineal gland

Summary In the present study the central innervation of the guinea-pig pineal gland was investigated. The habenulae and the pineal stalk contain myelinated and non-myelinated nerve fibres with few dense-cored and electron-lucent vesicles. Some myelinated fibres leave the main nerve fibre bundles, lose their myelin-sheaths and terminate in the pineal gland. Although direct proof is lacking, the non-myelinated fibres appear to end near the site where the bulk of the myelinated fibres are located. Here a neuropil area exists where synapses between non-myelinated fibre elements are abundant. Neurosecretory fibres were also seen. The results support the concept of functional interrelationships between hypothalamus, epithalamus and the pineal gland.     

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)