Summary
A technique is described for highly sensitive and precise visualization of central catecholamine systems in paraffin sections of freeze-dried tissue. The procedure is based on perfusion of the animal with a solution containing formaldehyde and/or glyoxylic acid, in the presence of a very high magnesium content (40 g MgSO4/150 ml solution) and acid pH. The perfused tissue is rapidly frozen, freeze-dried, treated with formaldehyde vapours (at +80°C for 1h), embedded in paraffin in vacuo, and finally sectioned.
The present technique has a sensitivity for the dopamine- and noradrenaline-containing systems that is comparable with that of the glyoxylic acid-Vibratome technique, which utilizes fresh, glyoxylic acid-perfused tissue. Thus, the preterminal axon pathways become fluorescent throughout their full extent and the several new terminal systems, discovered with the glyoxylic acid-Vibratome method, are well demonstrable. The method is also highly useful for the study of the cell bodies and their dendritic processes. The catacholamine fibre systems are visualized without any signs of diffusion and with a richness in detail. In animals pretreated with l-tryptophan and MAO-inhibitor the technique is also useful for studies on central indolamine-containing systems.
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References
Aghajanian, G.K., Kuhar, M.J., Roth, R.H.: Serotonin-containing neuronal perikarya and terminals: differential effects of p-chlorophenylalanine. Brain Res. 54, 85–101 (1973)
Björklund, A., Falck, B., Owman, Ch.: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In: Methods of investigative and diagnostic endocrinology, edit. by S.A. Berson, Vol. 1: The thyroid and biogenic amines (ed. J.E. Rall and I.J. Kopin, pp. 318–368. Amsterdam: North-Holland Publ. Comp. 1972
Björklund, A., Lindvall, O.: Dopamine in dendrites of substantia nigra neurons: suggestions for a role in dendritic terminals. Brain Res. 83, 531–537 (1975)
Björklund, A., Lindvall, O., Nobin, A.: Evidence of an incerto-hypothalamic dopamine neurone system in the rat. Brain Res. 89, 29–42 (1975)
Björklund, A., Nobin, A., Stenevi, U.: Acid catalysis of the formaldehyde condensation reaction for a sensitive histochemical demonstration of tryptamines and 3-methoxylated phenylethylamines. 2. Characterization of amine fluorophores and application to tissues. J. Histochem. Cytochem. 19, 286–298 (1971)
Björklund, A., Stenevi, U.: Acid catalysis of the formaldehyde condensation reaction for sensitive histochemical demonstration of tryptamines and 3-methoxylated phenylethylamines. 1. Model experiments. J. Histochem. Cytochem. 18, 794–802 (1970)
Bloom, F.F., Battenberg, E.L.F.: A rapid, simple and more sensitive method for the demonstration of central catecholamine-containing neurons and axons by glyoxylic acid induced fluorescence: II.: A detailed description of methodology. J. Histochem. Cytochem. 24, 561–571 (1976)
Boullin, D.J.: The action of extracellular cations on the release of the sympathetic transmitter from peripheral nerves. J. Physiol. (Lond.) 189, 85–99 (1967)
Carlsson, A., Hillarp, N.Å., Waldeck, B.: Analysis of the Mg++-ATP dependent storage mechanism in the amine granules of the adrenal medulla. Acta physiol. scand. 59, Suppl. 215, 1–38 (1963)
Eränkö, O., Räisänen, L.: Demonstration of catecholamines in adrenergic nerve fibres by fixation in aqueous formaldehyde solution and fluorescence microscopy. J. Histochem. Cytochem. 14, 690–691 (1966)
Farnebo, L.-O.: Release of monoamines evoked by field stimulation-studies on some ionic and metabolic requirements. Acta physiol. scand., Suppl. 371, 19–27 (1971)
Fuxe, K., Hamberger, B., Hökfelt, T.: Distribution of noradrenaline nerve terminals in cortical areas of the rat. Brain Res. 8, 128–131 (1968)
Fuxe, K., Jonsson, G.: A modification of the histochemical fluorescence method for the improved localization of 5-hydroxytryptamine. Histochemie 11, 161–166 (1967)
Hökfelt, T., Fuxe, K.: Cerebellar monoamine nerve terminals, a new type of afferent fibers to the cortex cerebelli. Exp. Brain Res. 9, 63–72 (1969)
Hökfelt, T., Ljungdahl, Å.: Modification of the Falck-Hillarp formaldehyde fluorescence method using the Vibratome®: simple, rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue. Histochemie 29, 325–339 (1972)
Kirpekar, S.M., Misu, Y.: Release of noradrenaline by splenic nerve stimulation and its dependence of calcium. J. Physiol. (Lond.) 188, 219–234 (1967)
Latis, A.M., Lund, R., Jacobowitz, D.: A simplified method for the histochemical localization of cardiac catecholamine-containing nerve fibers. J. Histochem. Cytochem. 15, 535–541 (1967)
Lindvall, O.: Mesencephalic dopaminergic afferents to the lateral septal nucleus of the rat. Brain Res. 87, 89–95 (1975)
Lindvall, O., Björklund, A.: The organization of the ascending catecholamine neuron systems in the rat brain. Acta physiol. scand., Suppl. 412, 1–48 (1974a)
Lindvall, O., Björklund, A.: The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistry 39, 97–127 (1974b)
Lindvall, O., Björklund, A., Hökfelt, T., Ljungdahl, Å.: Application of the glyoxylic acid method to Vibratome sections for improved visualization of central catecholamine neurons. Histochemie 35, 31–38 (1973)
Lindvall, O., Björklund, A., Nobin, A., Stenevi, U.: The adrenergic innervation of the rat thalamus as revealed by the glyoxylic acid fluorescence method. J. comp. Neurol. 154, 317–437 (1974a)
Lindvall, O., Björklund, A., Moore, R.Y., Stenevi, U.: Mesencephalic dopamine neurons projecting to neocortex. Brain Res. 81, 325–331 (1974b)
Lindvall, O., Björklund, A., Svensson, L.-Å.: Fluorophore formation from catecholamines and related compounds in the glyoxylic acid fluorescence histochemical method. Histochemistry 39, 197–227 (1974c)
Lorén, I., Björklund, A., Lindvall, O.: Improved visualization of developing catecholamine neurons in the CNS. To be published (1976)
Pease, D.C.: Buffered formaldehyde as a killing agent and primary fixative for electron microscopy. Anat. Rec. 142, 342 (1962)
Philippu, A.: Transport in intraneuronal storage vesicles. In: The mechanism of neuronal and extraneuronal transport of catecholamines, edit. by D.M. Paton, pp. 215–247. New York: Raven Press 1976
Svensson, L.-Å., Björklund, A., Lindvall, O.: Studies of the fluorophore forming reactions of various catecholamines and tetrahydroisoquinolines with glyoxylic acid. Acta chem. scand. B29, 341–348 (1975)
Watson, S.J., Barchas, J.D., Ellison, J.P.: Cryostat technique for central nervous system histofluorescence. Histochemistry (in press), 1976
Whaley, W.M., Govindachari, T.R.: The Pictet-Spengler synthesis of tetrahydroisoquinolines and related compounds. In: Organic reactions. Coll. Vol. 6, edit. by R. Adams, pp. 151–206. New York: John Wiley and Sons, Inc. 1951
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Lorén, I., Björklund, A., Falck, B. et al. An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH. Histochemistry 49, 177–192 (1976). https://doi.org/10.1007/BF00492374
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DOI: https://doi.org/10.1007/BF00492374