Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro

Summary In parallel fine structural, fluorescence histochemical and biochemical experiments the effect of 6-OH-DA administered in vivo and in vitro on the adrenergic nerves in the mouse iris was studied. As seen in the electron microscope, in vivo administration of 6-OH-DA causes a selective, rapid degeneration of the adrenergic axon terminals similar to that found after axotomy, whereas the cholinergic nerves are unaffected at all time intervals studied. Already 1 hr after the injection of 6-OH-DA the axonal enlargements swell and the size of the dense core of the granular vesicles is strongly reduced. Since the NA stores are almost completely depleted at this time interval, the small core present may be due to a reaction between 6-OH-DA and the fixative. From 2–4 hr after the injection increasing numbers of axonal enlargements with a high electron density are observed in the Schwann cell cytoplasm, which later are digested and completely absent about 48–72 hr after the 6-OH-DA injection. During the following weeks adrenergic axons reappear. This time course of degeneration obtained is considerably faster than that seen after axotomy in other studies. After incubation in 6-OH-DA containing media similar changes were observed in the axonal enlargements, starting already after 30 min of incubation. At this time-point there is a considerable reduction of endogenous NA and a severe damage of the membrane pump uptake mechanism. Incubation with 6-OH-DA and subsequent rinsing for 2 hr caused marked changes, including partly swelling of axons and partly shrinking of the axons into electron dense bodies.The fluorescence histochemical and biochemical results are in good agreement with the ultrastructural studies demonstrating a rapid loss of NA from the adrenergic nerve terminals and main axons and a long lasting depletion of the NA, with a gradual recovery to 75% 6 weeks after the injection.The investigation has been supported by research grants from the Swedish Medical Research Council (14X-2295, 14X-2887 and 04X-3881) Karolinska Institutet, Magnus Bergvalls and Carl-Berthel Nathorst Stiftelser. For generous supplies of drugs we are indebted to the following companies: AB Hässle (6-OH-DA, through Dr H. Corrodi), Pfizer (Niamid^®), Ciba (Serpasil^®). The skilful technical assistance of Miss Bodil Flock, Mrs Waltraut Hiort and Mrs Eva Lindqvist is gratefully acknowledged.     

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.     

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.     

Changes induced by 6-hydroxydopamine in the paraventricular nucleus

Summary Remodelling of catecholaminergic (CA) fibers after cerebral intraventricular 6-hydroxydopamine (6-OH-DA) administration was evaluated quantitatively in the paraventricular nucleus (PAR) of young adult rats, using fluorescence microscopy (FM) and electron microscopy (EM). Fluorescent CA varicosities and CA boutons (marked with 5-OH-DA) were counted after survival periods of 4, 21, 56 or 180 days. Four days after 6-OH-DA treatment, the number of fluorescent varicosities dropped to 45% of control numbers but was restored to 79% of control values by 180 days. In the EM study, marked boutons had dropped more dramatically: to 12% of control numbers, after 4 days and 54% by 180 days post-neurotoxin. These data provide strong evidence that substantial but incomplete restoration of CA terminals occurred in PAR. It is of interest that, in all survival intervals, percentage reductions in numbers of CA terminals were more extreme when EM was used for quantification. Nevertheless, the trends indicating partial restoration of terminal numbers with time were parallel in the FM and EM studies. Structures identified as CA growth cones in PAR contained a feltwork of fine filaments together with mitochondria, granular vesicles (often with electron-dense cores marked by the 5-OH-DA label), vacuoles and smooth-surfaced reticulum. The presence of growth cones, some of which persisted 11 months after neurotoxin administration, further supports the inference that a regenerative response of CA elements was evoked in PAR by the 6-OH-DA treatment.Presented in part at IV International Catecholamine Symposium in California, September 1978     

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     

Distribution of zinc in the substantia nigra of rats treated with 6-hydroxydopamine

To study the relationship between tissue accumulation of Zinc (Zn) and neurodegeneration in the nigrostriatal dopaminergic pathway,⁶⁵Zn distribution in this pathway was examined after unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rats. When⁶⁵ZnCl₂ was intravenously injected 4 days after treatment with 6-OHDA,⁶⁵Zn was concentrated in the ipsilateral substantia nigra 6 days after⁶⁵Zn injection. On the other hand, 19 d after treatment with 6-OHDA,⁶⁵Zn distribution in the ipsilateral substantia nigra was decreased to the level of the contralateral one. When NH₄ ⁹⁹TcO₄, which cannot go through the blood-brain barrier, was injected into rats 4 d after treatment with 6-OHDA,⁹⁹Tc was concentrated in the ipsilateral substantia nigra 30 min after⁹⁹Tc injection, but no longer detectable 6 d after injection. These results suggest that Zn is necessary for a repair process called replacement gliosis after the death of neurons and that excess Zn does not accumulate in the lesion after completion of the gliosis.     

Neuroblastoma cells expressing the noradrenaline transporter are destroyed more selectively by 6-fluorodopamine than by 6-hydroxydopamine

6-Hydroxydopamine (6-OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6-OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6-OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6-fluorodopamine (6-FDA) hydrolyzes slowly to 6-OHDA at physiological pH. Oxygen consumption, H(2)O(2), and quinone production are found to be intermediate between those of 6-OHDA and dopamine (DA). Relative neurotoxicity of these compounds was assessed by cell viability and DNA damage in the human neuroblastoma lines SH-SY5Y and SK-N-LO, which express and lack the noradrenaline transporter, respectively. Specific uptake of DA and 6-FDA by SH-SY5Y cells was demonstrated by competitive m-[(131)I]iodobenzylguanidine uptake inhibition. The competition by 6-OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6-FDA toxicity was preferential for SH-SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6-FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6-OHDA cytotoxicity.     

Pharmacomorphological study of denervation induced by 6-hydroxydopamine in Porichthys photophores

The effects of 6-hydroxydopamine (6-OHDA) on the bioluminescent response of Porichthys photophores were investigated as part of a pharmacological study of the neural control of luminescence in this fish. Subcutaneous injections of 6-OHDA induce a luminescent response similar to that of norepinephrine (NE), suggesting a sympathomimetic action. The luminescent response to electrical stimulation is almost completely and irreversibly abolished within 24 hours following low-dose treatment of the photophores with 6-OHDA, while the sensitivity of these organs to exogenous NE is increased significantly over the few days post-treatment. During this period the photophores continuously emitted a steady low-level glow. Electronmicroscopic studies of such photophores revealed progressive destruction of the nerve endings. Photophore luminescent sensitivity to NE subsequently became sub-normal, and at this stage electron microscopy revealed an increasingly larger number of damaged photocytes, supportive cells and, in one case, lens cells. From these results it is suggested that 6-OHDA initially impairs neuro-photocyte transmission by destroying catecholaminergic nerve endings. In turn, the transmitter reuptake mechanism is also impaired, thus accounting for development of supersensitive responses to exogenous NE. Subnormal luminescent responses to NE appear as a result of loss of photocyte competence due to structural deterioration. The latter are interpreted as the consequence of removal of trophic factors supplied by the photophore adrenergic innervation.Suppression of luminescent response to both electrical stimulation and exogenous NE in photophores treated with higher doses of 6-OHDA, may be due to a direct effect of this drug on the receptor sites of the photocytes.     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

A comparative study of the adrenergic nerves to the anterior eye segment of some primates

Summary The distribution of adrenergic terminals to the anterior eye segment of humans, Cynomolgue monkeys, squirrel monkeys, owl monkeys, Cebus monkeys, vervets, tamarins, and baboons has been investigated. The cornea is normally devoid of adrenergic terminals, except in a plexus near the limbus. The trabecular meshwork contains varying numbers of adrenergic terminals: usually none in Cynomolgus monkeys, patas monkeys, vervets, and humans, although fibres have very rarely been observed in Cynomolgus monkeys, vervets, and humans; a few in owl monkeys, squirrel monkeys, and tamarins; and moderate numbers in Cebus monkeys and baboons. From the evidence, however, it seems premature to presume an adrenergic innervation of the trabecular mechanism regulating the outflow resistance. The dilatator pupillae is regularly supplied with numerous adrenergic terminals and in the iris stroma there is probably an adrenergic innervation of the melanophores. The sphincter pupillae regularly contains adrenergic terminals with notable species differences; most fibres occur in baboons and fewest in humans, with the remaining species forming a middle class. The ciliary processes in all species contain a moderate number of adrenergic terminals, presumably primarily associated with the epithelium. Intraepithelial adrenergic terminals have been observed on the pars plana of the ciliary body of humans, Cebus monkeys, vervets, baboons, and patas monkeys. The ciliary muscle of baboons and Cynomolgus monkeys contains numerous adrenergic terminals. Moderate numbers occur in Cebus monkeys and vervets, and still less in (in falling order) tamarins, squirrel monkeys, humans, and patas monkeys.     

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.     

Extraneuronal effects of 6-hydroxydopamine

Summary The effects of various concentrations of 6-hydroxydopamine (6OHDA) on rat adrenocortical cells in tissue culture were studied with phase contrast and electron microscopy. With 40 mg/l of 6-OHDA the first signs of alteration as revealed by microcinematography appeared in isolated cortical cells as early as 15 min after addition of the drug. There was a cessation of movement of cell organelles and an immobilisation of membrane undulations followed by the development of dark inclusion bodies. The cells underwent increasing shrinkage and collapsed by 11/2 h. Chromaffin cells were not affected until 45 min after exposure to the drug and neurons were the most resistant population. However 61/2 h after application of the drug most cells in the culture were dead. 6-OHDA applied in different doses and to adrenal expiants did not alter the sequence of events. Ultrastructurally cortex cells underwent damage along two lines: they either showed lytic changes or developed various types of dense bodies before reaching the lytic stage.Treatment of cortical cells with 40 mg/l 5-or 6-OHDA followed by exposure to buffered 2% glyoxylic acid and heat did not produce a fluorescence within the cells. Microspectrofluorimetry on amine models with noradrenaline, 5- and 6-OHDA revealed that neither 5-nor 6-OHDA are capable to form a fluorophore with glyoxylic acid.Dedicated to Professor Berta Scharrer in honor of her 70th birthdaySupported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellowship of the University of Melbourne to K.U., and a Research Fellowship and Grant-in-Aid from the Life Insurance Medical Research Fund of Australia and New Zealand to J.H.C.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

Overhauser-enhanced magnetic resonance imaging characterization of mitochondria functional changes in the 6-hydroxydopamine rat model

Oxidative stress may be involved in the dopaminergic neurodegenerations seen in 6-OHDA-lesioned rats through its production of free radicals and through mitochondrial dysfunction. In this study, we noninvasively demonstrate brain redox alterations in 6-OHDA-lesioned rats using Overhauser-enhanced magnetic resonance imaging (OMRI). The reduction rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-l-oxyl (methoxycarbonyl-PROXYL), a redox-sensitive contrast agent, was used as an index of the redox status in vivo. The methoxycarbonyl-PROXYL reduction rate, calculated from continuous images, decreased significantly in lesioned hemispheres compared to their corresponding contralateral hemispheres. The reduction rates in cellular fractions obtained from the striatum were estimated by X-band electron spin resonance (ESR) and calculated by assuming first-order kinetics for their time-dependent decreases. When methoxycarbonyl-PROXYL was mixed with cytoplasm fractions, the reduction rates were the same in both hemispheres. However, the ESR signal of methoxycarbonyl-PROXYL in the mitochondrial fraction of the lesioned hemispheres decayed more slowly than that of the corresponding contralateral hemispheres. Concordantly, biochemical assays showed that the activity of mitochondrial complex I also decreased more slowly in lesioned hemispheres. Thus, this method of noninvasively imaging brain redox alterations faithfully reflects changes in mitochondrial complex I activity in 6-OHDA-lesioned rats.     

Adrenergic innervation of pancreatic islets and modulation of insulin secretion by the sympatho-adrenal system

Summary Morphological changes in the adrenergic innervation of pancreatic islets after chemical sympathectomy by use of 6-hydroxydopamine and the influence of the sympatho-adrenal system on insulin secretion were investigated in the mouse and rat.Fluorescence histochemistry revealed a clear-cut reduction in the number of adrenergic nerve fibers in the pancreatic islets 2 days after administration of 6-hydroxydopamine; the reduction was more pronounced in the rat than in the mouse. In the rat, a partial regeneration was seen after 6 weeks. In the pancreas of the mouse, after administration of 6-hydroxydopamine, a severe damage of unmyelinated nerve fibers was revealed electron microscopically. However, no ultrastructural or immunohistochemical alterations could be demonstrated in the endocrine cells of the islets.6-Hydroxydopamine induced a depression of basal plasma insulin concentrations in mice and an elevation in rats. Adrenalectomy depressed basal plasma insulin levels in mice.The -adrenoceptor antagonist phentolamine enhanced insulin secretion in normal mice. The secretory response of insulin to phentolamine was diminished by chemical sympathectomy and almost abolished by adrenalectomy or the combination of chemical sympathectomy and adrenalectomy. Thus, the effect of phentolamine is probably mediated by liberated catecholamines.It is concluded that basal insulin secretion is partially regulated by the sympatho-adrenal system and that species differences exist in this respect. In addition, the results suggest that endogenous catecholamines have the ability to promote insulin secretion.