A fluorescence histochemical study of the degeneration and regeneration of noradrenergic nerves in the chick following treatment with 6-hydroxydopamine

Summary The fluorescence histochemical method of Falck and Hillarp for the cellular localisation of biogenic amines was used to follow the disappearance and reappearance of peripheral noradrenergic nerves in the chick after treatment with 6-hydroxydopamine. With certain exceptions, it seemed that the disappearance and reappearance of the nerves was due to their degeneration and subsequent regeneration. However, there was not a uniform destruction of the noradrenergic nerves associated with different effector tissues. The extent of degeneration was greater in older than in younger chicks following equivalent doses of the drug, possibly indicating a greater uptake efficiency of the older nerves. It appeared that the noradrenergic nerves in the younger chicks regenerated more rapidly than those of older chicks, but in none of the animals studied were there any obvious morphological modifications of the regenerating nerves.The major part of this work was carried out in the Department of Zoology, Melbourne University, Australia.We are grateful to Professor G. Burnstock for use of laboratory facilities, and to the National Heart Foundation of Australia for financial support.     

Fluorescent histochemical studies on the effects of 6-hydroxydopamine on adrenaline-containing nerves in the toad

Summary Fluorescence histochemistry has been used to study the effects of 6-hydroxydopamine (6-OHDA) (100 mg/kg injected into the dorsal lymph sac) on adrenaline-containing nerves in the large intestine, mesentery, lung, bladder and heart atria of the toad Bufo marinus. A gradual decrease both in fluorescence intensity and in number of detectable fibres during the first 4 hours after 6-OHDA was accompanied by a build-up of fluorescence in the nonterminal regions. These phenomena have been discussed in relation to the time course of the degeneration produced by 6-OHDA in noradrenergic nerves of higher vertebrates. Almost complete chemical sympathectomy was seen after one day, and it was not till 13 days that regenerating nerve fibres were seen in any organ. In the large intestine, however, re-innervation was slower, being first noted after 39 days. The time course of regeneration has been compared with that following sympathectomy in various mammalian organs.     

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.     

Fine structure and innervation of the avian adrenal gland

Summary Apart from cholinergic nerve fibers, which make up the main part of efferent fibers to the avian adrenal gland (Unsicker, 1973b), adrenergic, purinergic and afferent nerve fibers occur. Adrenergic nerve fibers are much more rare than cholinergic fibers. With the Falck-Hillarp fluorescence method they can be demonstrated in the capsule of the gland, in the pericapsular tissue and near blood vessels. By their green fluorescent varicosities they may be distinguished characteristically from undulating yellow fluorescent ramifications of small nerve cells which are found in the ganglia of the adrenal gland and below the capsule. The varicosities of adrenergic axons exhibit small (450 to 700 Å in diameter) and large (900 to 1300 Å in diameter) granular vesicles with a dense core which is usually situated excentrically. After the application of 6-hydroxydopamine degenerative changes appear in the varicosities. Adrenergic axons are not confined to blood vessels but can be found as well in close proximity of chromaffin cells. Probably adrenergic fibers are the axons of large ganglion cells which are situated mainly within the ganglia of the adrenal gland and in the periphery of the organ and whose dendritic endings show small granular vesicles after treatment with 6-OHDA.A third type of nerve fiber is characterized by varicosities containing dense-cored vesicles with a thin light halo, the mean diameter (1250 Å) of which exceeds that of the morphologically similar granular vesicles in cholinergic synapses. Those fibers resemble neurosecretory and purinergic axons and are therefore called p-type fibers. They cannot be stained with chromalum-hematoxyline-phloxine. Axon dilations showing aggregates of mitochondria, myelin bodies and dense-cored vesicles of different shape and diameter are considered to be afferent nerve endings. Blood vessels in the capsule of the gland are innervated by both cholinergic and adrenergic fibers.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1).     

Fluorescence histochemical and ultrastructural observations on the effects of intravenous injections of vinblastine on noradrenergic nerves

Summary Fluorescence histochemical and ultrastructural observations were made on noradrenergic nerves in chicks, following intravenous injections of vinblastine. The drug caused a reversible blockade of the axoplasmic transport of catecholamines in non-terminal axons. Some terminal fibres showed a loss of fluorescence 48 hours after injections of animals with 10 mg/kg vinblastine, and ultrastructurally there were signs of neural necrosis.Some of the work described in this paper was carried out in the Department of Zoology, Melbourne University. We are grateful to professor G. Burnstock for use of facilities, and the National Heart Foundation of Australia for financial assistance.     

The adrenergic innervation of the renal artery and vein of the rat

Summary The adrenergic innervation of the extrarenal blood vessels of the rat left kidney was investigated by fluorescence histochemistry and by electron microscopy. The trunk of the renal artery proximal to the aorta is elastic and appears to be very sparsely innervated. In contrast, near the kidney the renal artery—which divides into 3 to 4 large branches of the muscular type possesses a dense adrenergic innervation. The adrenergic terminal axons are situated in the adventitia close to the external elastic lamella, but only rarely in close contact with smooth muscle cells. In most instances several terminal axons are grouped and enclosed by a Schwann cell, single axons being rare. All terminal axons are able to take up and to store 5-hydroxydopamine which strongly suggests that they are adrenergic. The innervation of the renal vein is more sparse than that of the muscular arteries but somewhat denser than that of the elastic artery. In addition, close to the origin of the renal artery the presence of small intensively fluorescent (SIF) cells as well as of some adrenergic ganglion cells is noted. The latter are situated in the adrenergic nonterminal axon bundles, which run parallel to the blood vessels.It is concluded that the uneven adrenergic innervation along the artery as well as individual variations in the branching of the artery are the main causes of the unusually high individual variations of the NA content of this organ such as used in pharmacological experiments.     

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.     

Surface morphology of rat peritoneal mast cells during in vitro regeneration after histamine secretion

Summary Cell-surface morphology of regenerating mast cells was followed over a period of 48 h after histamine release. Control cells (not stimulated to secrete) were characterized by anastomosing folds of membrane of equal depth and width. During exocytosis these folds disappeared and were replaced by deep cup-shaped flaps of membrane evident in cells incubated for 10 min. During the first hours of regeneration these flaps fused mutually or with the plasma membrane. This activity suggests membrane retrieval, maybe specifically recycling the granule-type patches of membrane. Membrane-fusion activity was observed to some degree also after extended incubation. After 48 h of incubation the regeneration process was still not completed, as indicated by the fact that holes leading to intracellular cavities could still be found.     

Effects of systemic administration of 6-hydroxydopamine on the circumventricular organs in nonhuman primates

Summary The neurotoxin 6-hydroxydopamine (6-OH-DA) has been shown to produces degenerative changes in noradrenergic nerve terminals and preterminals in the CNS following intracisternal, intraventricular, and stereotaxic injection into the brain parenchyma. Systemic injections of this drug are also known to result in degenerative changes in noradrenergic terminals in the peripheral nervous system and in the circumventricular organs (CVO; areas of the CNS which lie outside the blood brain barrier). In the present study eight adult female cynomolgus monkeys were employed. The four experimental animals were injected on two successive days with 150 and 200 mg/kg 6-OH-DA, respectively. The four controls received only the diluent consisting of 0.1% ascorbic acid in normal saline. Two animals from each of the experimental and control groups were sacrificed at 2 h and 24 h after the second injection. Degenerative changes in the SFO neurons were characterized by a generalized increase in electron density of cytoplasmic elements in axonal terminals and preterminals. Multilamellar bodies, and increases in the number of dense core vesicles, dense bodies and secondary lysosomes were also observed after treatment with 6-OH-DA. The neurons showed clumping of mitochondria, which also appeared to be undergoing degenerative changes. The vacuoles in some supraependymal cells were greatly dilated as was the Golgi apparatus in the ependymal cells. The ependymal cell layer appeared to be intact, but there were areas immediately deep to this cell layer that contained large extracellular spaces. This increase in extracellular space was also commonly observed surrounding the perivascular spaces. These phenomena greatly contribute to the spongy appearance that the SFO takes on after 6-OH-DA administration.Supported by: NASA-Ames NSG-2139 and NIH RR00164-16     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Galanin-immunoreactive nerves in the mouse and rat pancreas

Summary Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin-immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non-adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas.     

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.     

The synthesis of NPY and DBH is independently regulated in adrenergic nerves after reserpine

A newly developed cytofluorimetric scanning technique was applied in a pharmacological study to investigate the influence of reserpine (10 mg/kg) on the axonal transport of norepinephrine (NE), dopamine--hydroxylase (DBH), tyrosine hydroxylase (TH), and neuropeptide Y (NPY)-like immunoreactivities (LI) in the adrenergic axons of the sciatic nerve of rat. Early after reserpine (18 hr and 24 hr after the reserpine injection) the amounts of NE accumulated proximal to a 12-hr crush werenil or very low, as observed in earlier studies. DBH-LI, TH-LI, and NPY-LI accumulations were also depressed but only to about 50% of control accumulations. This decrease in amounts of transported substances was probably caused by a decrease in protein synthesis and also a lowered velocity of fast axonal transport initially after reserpine, when body temperature is low. The amounts of accumulated NE, DBH-LI, TH-LI, and NPY-LI were normalized around day 2 after reserpine, but on day 4 NE, DBH-LI, and in some rats also TH-LI accumulated in supranormal amounts. However, NPY-LI accumulations were normal, indicating that DBH, butrot NPY, was trans- synaptically induced in rat sympathetic neurons, and that the biochemical composition of axonally transported organelles is altered for some days after reserpine.Dedicated to Dr. Abel Lajtha.     

Fine structure of baroreceptor terminals in the carotid sinus of guinea pigs and mice

Summary A light and electron microscopic study was undertaken on the baroreceptor axon terminals in the carotid sinus of guinea pigs and mice, using serial semithin and thin sections.Together with their enveloping Schwann cells, numerous lanceolate axon terminals are organized into a well-defined discoid end organ, referred to as the baroreceptor unit. Baroreceptor units measure 100 to 150 m in diameter and are arranged in a hexagonal pattern. These end organs represent free branched lanceolate mechanoreceptors of complex type (Andres and von Düring, 1973) which belong to the main group of stretch receptors.In the guinea pig the lanceolate terminals enter the media and approach the innermost layers near the intima. In the mouse the terminals are seen to spread in the adventitia and along the medio-adventitial border. Only a few of them penetrate the external elastic layer. Species differences concerning the localization and extent of these visceral mechanoreceptors are discussed, as well as the modified architecture of the sinus wall in the receptor area (elastic segment).Lanceolate terminals form beaded varicosities which are equipped with finger-like or lamellar axoplasmic protrusions. These projections contain a well-differentiated receptor matrix. They are attached to collagen and elastic fibers. The varicosities include densely packed mitochondria, neurotubules, profiles of axoplasmic reticulum, clear and granular vesicles, and striking accumulations of glycogen particles, lamellated bodies and lysosomes. Four types of varicosities are discerned according to their main axoplasmic components. Various types of these varicosities occur within an individual lanceolate terminal.The adrenergic innervation of the carotid sinus was studied by fluorescence histochemistry. In guinea pigs a multilayered wide-meshed plexus of fluorescent fibers occurs in the adventitia where it is closely related to baroreceptor stem fibers. However, adrenergic axons do not enter the media. In mice fluorescent fibers are extremely rare in the adventitia of the carotid sinus.Dedicated to Prof. Dr. Drs.h.c. W. Bargmann on the occasion of his 70th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft Nr. Bo/525-1. These results were presented in part at the 17. Tagung der Deutschen Gesellschaft für Elektronenmikroskopie, Berlin, Sept. 21.–26., 1975     

Vasomotor innervation in the hind limb muscles of the normal and 6-hydroxydopamine treated cat

Summary Vasomotor nervous distribution to arteries in the cat gastrocnemius and soleus muscles was investigated in the normal and 6-hydroxydopamine treated animal. Muscle samples were processed for the demonstration of acetylcholinesterase by a thiocholine method and for the presence of catecholamines by a formol fluorescence technique. In isolated perfused muscle preparations noradrenaline or adrenaline increased perfusion pressure while acetylcholine or isoprenaline decreased this pressure. A beaded fluorescent periarterial plexus (not visualised after reserpine treatment) was observed. Vasomotor axons, all of which were ACHE-negative, exhibited degenerative changes following 6-hydroxydopamine injection, while ACHE-positive axons outside the adventitial sheath were unaffected. No indication of cholinergic periarterial nerves was found and our evidence suggests that the vasomotor innervation is adrenergic sympathetic in nature.This work was supported by a grant from the Medical Research Council. During the tenure of a Wellcome Trust Fellowship, Dr. F. Joó is on leave of absence from Jozsef Attila University, Szeged, Hungary. The electron microscope is on permanent loan to one of us (J. D. L.) from the Wellcome Trust. We are most grateful to Mrs. G. Howells and Miss D. Morgan for technical assistance and to Mr. P. Hire for photographic help.