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.
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References
Alksne, J.F., Blackstad, T.W., Walberg, F., White, L.E., Jr.: Electron microscopy of axon degeneration: A valuable tool in experimental neuroanatomy. Ergebn. Anat. Entwickl.-Gesch. 39, 1–32 (1966).
Angeletti, P.U., Levi-Montalcini, R.: Sympathetic nerve cell destruction in newborn mammals by 6-hydroxydopamine. Proc. Nat. Acad. Sci. (Wash.) 65, 114–121 (1970).
Ausprunk, D.H., Berman, H., McNary, Wm.F., Jr.: Perivascular nerve degeneration produced in the hamster cheek pouch by 6-hydroxydopamine. Europ. J. Pharmacol. 15, 292–299 (1971).
Bennett, T., Burnstock, G., Cobb, J.L.S., Malmfors, T.: An ultrastructural and histochemical study of the short-term effects of 6-hydroxydopamine on adrenergic nerves in the domestic fowl. Brit. J. Pharmacol. 38, 802–809 (1970).
Cheah, T.B., Geffen, L.B., Jarrott, B., Ostberg, A.: Action of 6-hydroxydopamine on lamb sympathetic ganglia, vas deferens and adrenal medulla: a combined histochemical, ultrastructural and biochemical comparison with the effects of reserpine. Brit. J. Pharmacol. 42, 543–557 (1971).
Corrodi, H., Jonsson, G.: The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem. 15, 65–78 (1967).
Dahlström, A.: The intraneuronal distribution of noradrenaline and the transport and lifespan of amine storage granules in the sympathetic adrenergic neuron. M. D. Thesis, Stockholm 1966.
Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta physiol. scand. 56, Suppl. 197 (1962).
Fleming, R.M., Clark, W.G.: Quantitative thin-layer chromatographic estimation of labeled dopamine and norepinephrine, their precursors and metabolites. J. Chromatog. 52, 305–312 (1970).
Furness, J.B., Campbell, G.R., Gillard, S.M., Malmfors, T., Cobb J.L.S., Burnstock, G.: Cellular studies of sympathectic denervation produced by 6-hydroxydopamine in the vas deferens. J. Pharmacol. exp. Ther. 174, 111–122 (1970).
Goldman, H., Jacobowitz, D.: Correlation of norepinephrine content with observations of adrenergic nerves after a single dose of 6-hydroxydopamine in the rat. J. Pharmacol. exp. Ther. 176, 119–133 (1971).
Grillo, M., Palay, S.L.: Granule-containing vesicles in the autonomic nervous system. In: Electron microscopy (ed.: S.S. Breese, Jr.), 2, U-1. New York: Academic Press 1962.
Haeusler, G.: Early pre- and postjunctional effects of 6-hydroxydopamine. J. Pharmacol. exp. Ther. 178, 49–62 (1971).
Hamberger, B.: Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. Acta physiol. scand., Suppl. 295 (1967).
Hámori, J., Lang, E., Simon, L.: Experimental degeneration of the preganglionic fibers in the superior cervical ganglion of the cat. Z. Zellforsch. 90, 37–52 (1968).
Hökfelt, T.: In vitro studies on central and peripheral monoamine neurons at the ultrastructural level. Z. Zellforsch. 91, 1–74 (1968).
Hökfelt, T.: Distribution of noradrenaline storing particles in peripheral adrenergic neurons as revealed by electron microscopy. Acta physiol. scand. 76, 427–440 (1969).
Hökfelt, T., Ungerstedt, U.: Electron and fluorescence microscopical studies on the nucleus caudatus putamen of the rat after unilateral lesions of ascending nigro-neostriatal dopamine neurons. Acta physiol. scand. 76, 415–426 (1969).
Hökfelt, T., Ungerstedt, U.: Effects of 6-hydroxydopamine on central monoamine neurons with special reference to the nigro-striatal dopamine systems: An electron and fluorescence microscopical study. (To be published.)
Iwayama, T.: Ultrastructural changes in the nerves innervating the cerebral artery after sympathectomy. Z. Zellforsch. 109, 465–480 (1970).
Jaim-Etcheverry, G., Zieher, L.M.: Permanent depletion of peripheral norepinephrine in rats treated at birth with 6-hydroxydopamine. Europ. J. Pharmacol. 13, 272–276 (1971).
Jonsson, G., Hamberger, B., Malmfors, T., Sachs, Ch.: Uptake and accumulation of 3H-noradrenaline in adrenergic nerves of rat iris-effect of reserpine, monoamine-oxidase and tyrosine-hydroxylase inhibition. Europ. J. Pharmacol. 8, 58–72 (1969).
Jonsson, G., Malmfors, T.: Determinations of small amounts of noradrenaline using a fluorescence hitsochemical method. Histochemie 24, 1–6 (1970).
Jonsson, G., Sachs, Ch.: Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons. Europ. J. Pharmacol. 9, 141–155 (1970).
Jonsson, G., Sachs, Ch.: Degenerative and nondegenerative effects of 6-hydroxydopamine on adrenergic nerves. J. Pharmacol. exp. Ther. 180, 625–635 (1972).
Knyihár, F., Ristovsky, K., Kalman, K.G., Csillik, B.: Chemical sympathectomy: Histochemical and submicroscopical consequences of 6-hydroxydopamine treatment in the rat iris. Experientia (Basel) 25, 518–520 (1969).
Luft, J.H.: Permanganate—a new fixative for electron microscopy. J. biophys. biochem. Cytol. 2, 799–802 (1956).
Luft, J.H.: Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409–414 (1961).
Malmfors, T.: Studies on adrenergic nerves. Acta physiol. scand. 64, Suppl. 248 (1965).
Malmfors, T., Sachs, Ch.: Direct studies on the disappearance of the transmitter and changes in the uptake-storage mechanisms of degenerating adrenergic nerves. Acta physiol. scand. 64, 211–223 (1965).
Malmfors, T., Sachs, Ch.: Degeneration of adrenergic nerves produced by 6-hydroxydopamine. Europ. J. Pharmacol. 3, 89–92 (1968).
Olson, L., Malmfors, T.: Growth characteristics of adrenergic nerves in the adult rat. Acta physiol. scand., Suppl. 348 (1970).
Orden, III, L.S. van, Bensch, K.G., Langer, S.Z., Trendelenburg, U.: Histochemical and fine structural aspects of the onset of denervation supersensitivity in the nictitating membrane of the spinal cat. J. Pharmacol. exp. Ther. 157, 274–283 (1967).
Porter, C.C., Totaro, J.A., Stone, C.A.: Effect of 6-hydroxydopamine and some other compounds on the concentration of norepinephrine in the hearts of mice. J. Pharmacol. exp. Ther. 140, 308–316 (1963).
Richardson, K.C.: Electron microscopic identification of autonomic nerve endings. Nature (Lond.) 210, 756 (1966).
Roth, C.D., Richardson, K.C.: Electron microscopical studies on axonal degeneration in the rat iris following ganglionectomy. Amer. J. Anat. 124, 341–360 (1968).
Sachs, Ch.: Noradrenaline uptake mechanisms in the mouse atrium. Acta physiol. scand., Suppl. 341 (1970).
Sachs, Ch., Champlain, J. de, Malmfors, T., Olson, L.: The postnatal development of noradrenaline uptake in the adrenergic nerves of different tissues from the rat. Europ. J. Pharmacol. 9, 67–79 (1970).
Saelens, J.K., Schoen, M.S., Kovacsics, G.B.: An enzyme assay for norepinephrine in brain tissue. Biochem. Pharmacol. 16, 1043–1049 (1967).
Siggins, G.R., Bloom, F.E.: Cytochemical and physiological effects of 6-hydroxydopamine on periarteriolar nerves of frogs. Circulat. Res. 27, 23–38 (1970).
Stone, C.A., Stavorski, J.M., Ludden, C.T., Wenger, H.C., Ross, C.A., Totaro, J.A., Porter, C.C.: Comparison of some pharmacologic effects of certain 6-substituted dopamine derivatives with reserpine, guanethidine and metaraminol. J. Pharmacol. exp. Ther. 142, 147–156 (1963).
Thoenen, H., Tranzer, J.P.: Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 261, 271–288 (1968).
Tranzer, J.P., Richards, J.G.: Fine structural aspects of the effect of 6-Hydroxydopamine on peripheral adrenergic neurons. In: 6-hydroxydopamine and catecholamine neurons (eds: T. Malmfors and H. Thoenen), p. 15–31. Amsterdam-London: North-Holland Publ. Comp. 1971.
Tranzer, J.P., Thoenen, H.: Ultramorphologische Veränderungen der sympathischen Nervenendigungen der Katze nach Vorbehandlung mit 5- und 6-Hydroxy-Dopamin. Naunyn-Schmiedebergs Arch. Pharmak. exp. Path. 257, 343 (1967a).
Tranzer, J.P., Thoenen, H.: Electronmicroscopic localization of 5-hydroxydopamine (3,4,5-trihydroxy-phenyl-ethylamine), a new “false” sympathetic transmitter. Experientia (Basel) 23, 743–745 (1967b).
Tranzer, J.P., Thoenen, H.: An electron microscopic study of selective, acute degeneration of sympathetic nerve terminals after administration of 6-hydroxydopamine. Experientia (Basel) 24, 155–156 (1968).
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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.
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Hökfelt, T., Jonsson, G. & Sachs, C. Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro . Z.Zellforsch 131, 529–543 (1972). https://doi.org/10.1007/BF00306969
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DOI: https://doi.org/10.1007/BF00306969