Summary
Combined light and electron microscopy of identified neurons requires an intracellular marker that is both photon opaque and has electron scattering properties. We describe results using cobalt chloride block intensified with silver as an intracellular label. The novelty of the method is its integration in tissue fixation, prior to dehydration, resulting in fine grain precipitates that resolve certain intracellular structures. Filled neurons are clearly distinguishable from unfilled profiles by cobalt-silver precipitates. Energy dispersive X-ray analysis confirms that silver is specifically deposited onto cobalt sulphide cores which are characteristically associated with microtubules, mitochondria, presynaptic and postsynaptic specializations and gap junction-like membrane appositions.
Similar content being viewed by others
References
Altman, J. S., Shaw, M. K. &Tyrer, N. M. (1979) Visualisation of synapses of physiologically identified cobalt-filled neurones in the locust.Journal of Physiology 296, 2–3P.
Altman, J. S. &Tyrer, N. M. (1980) Filling selected neurons with cobalt through cut axons. InNeuroanatomical Techniques. Insect Nervous System (edited byStrausfeld, N. J. &Miller, T. A.), pp. 373–402. New York, Heidelberg, Berlin: Springer Verlag.
Bacon, J. P. &Altman, J. S. (1977) A silver intensification method for cobalt-filled neurones in wholemount preparations.Brain Research 138, 359–63.
Bacon, J. &Strausfeld, N. J. (1980) Nonrandom resolution of neuron arrangements. InNeuroanatomical Techniques. Insect Nervous System (edited byStrausfeld, N. J. &Miller, T. A.), pp. 357–72. New York, Heidelberg, Berlin: Springer Verlag.
Bennett, M. V. L. (1972) Electrical transmission: a functional analysis and comparison to chemical transmission. InHandbook of Physiology, I, The Nervous System (edited byKandel, E. R.), pp. 357–416. Bethesda, Maryland: American Physiological Society.
Boschek, C. B. (1971) On the fine structure of the peripheral retina and lamina ganglionaris of the flyMusca domestica.Zeitschrift für Zellforschung und mikroskopische Anatomie 118, 369–409.
Brightman, M. W. &Reese, T. S. (1969) Junctions between intimately apposed cell membranes in the vertebrate brain.Journal of Cell Biology 40, 648–77.
Carafoli, E., Gazzotti, P., Schwerzmann, K. &Niggli, V. (1977) Mitochondrial calcium binding proteins. InCalcium-binding Proteins and Calcium function (edited byWasserman, R. H., Corradino, R. A., Carafoli, E., Kretsinger, R. H., Maclennan, D. H. &Siegel, F. L.), pp. 454–68. Amsterdam: Elsevier North Holland.
Carafoli, E. &Rossi, C. S. (1971) Calcium transport in mitochondria.Advances in Cytopharmacology 1, 209–27.
Carlin, R. K., Grab, D. J. &Siekevitz, P. (1981) Function of calmodulin in postsynaptic densities. III. Calmodulin binding proteins of the PSD.Journal of Cell Biology 89, 449–55.
Erulkar, S. D. &Fine, A. (1979) Calcium in the nervous system.Reviews of Neuroscience 4, 179–232.
Gilula, N. B. &Epstein, M. L. (1976) Cell-to-cell communication, gap junctions and calcium. InSymposia of the Society for Experimental Biology XXX: Calcium in Biological Systems (edited byDuncan, C. J.), pp. 257–72. Cambridge: The University Press.
Grab, D. J., Carlin, R. K. &Siekewitz, P. (1981) Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity.Journal of Cell Biology 89, 440–8.
Hackett, J. T. (1976) Selective antagonism of frog cerebellar synaptic transmission by manganese and cobalt ions.Brain Research 114, 47–52.
Hausen, K. &Wolburg-Buchholz, K. (1980) An improved cobalt sulfide-silver intensification method for electron microscopy.Brain Research 187, 462–6.
Hausen, K., Wolburg-Buchholz, K. &Ribi, W. A. (1980) The synaptic organisation of visual interneurons in the lobula complex of flies. A light and electron microscopical study using silver-intensified cobalt-impregnations.Cell and Tissue Research 208, 371–87.
Kita, H. &Van Der Kloot, W. (1973) Action of Co and Ni at the frog neuromuscular junction.Nature New Biology 245, 52–3.
Lane, N. J. (1978) Intercellular junctions and cell contacts in vertebrates. InProceedings of the Ninth International Congress of Electron Microscopy Vol. III (edited bySturgess, J. M.), pp. 673–91. Toronto: Microscopical Society of Canada.
Lane, N. J., Skaer, H. Le B. &Swales, L. S. (1977) Intercellular junctions in the central nervous system of insects.Journal of Cell Science 26, 175–99.
Llinas, R., Steinberg, I. Z. &Walton, K. (1976) Presynaptic calcium currents and their relation to synaptic transmission: Voltage clamp study in squid giant synapse and theoretical model for the calcium gate.Proceedings of the National Academy of Sciences USA 73, 2918.
Obermayer, M. &Strausfeld, N. J. (1980) Silver-staining cobalt sulfide deposits within neurons of intact ganglia. InNeuroanatomical Techniques. Insect Nervous System (edited byStrausfeld, N. J. &Miller, T. A.), pp. 403–27. New York, Heidelberg, Berlin: Springer Verlag.
Osborne, M. P. (1975) The ultrastructure of nerve-muscle synapses. InInsect Muscle (edited byUsherwood, P. N. R.), pp. 151–205. New York: Academic Press.
Peachey, L. D. (1964) Electron microscopic observations on the accumulation of divalent cations in intramitochondrial granules.Journal of Cell Biology 20, 95–108.
Peracchia, C. (1980) Structural correlates of gap junction permeation.International Review of Cytology 66, 81–146.
Pfenninger, K. H. (1971) The cytochemistry of synaptic densities. I. An analysis of the bismuth iodide impregnation method.Journal of Ultrastructure Research 34, 103–22.
Phillips, C. E. (1980) Intracellularly injected cobaltous ions accumulate at synaptic densities.Science 207, 1477–9.
Pitman, R. M., Tweedle, C. D. &Cohen, M. J. (1972) Branching of central neurons: Intracellular cobalt injection for light and electron microscopy.Science 176, 412–4.
Pitman, R. M., Tweedle, C. D. &Cohen, M. J. (1973) The form of nerve cells: determination by cobalt impregnation. InIntracellular Staining in Neurobiology (edited byKater, S. B. &Nicholson, C.), pp. 83–97. Berlin, New York: Springer Verlag.
Politoff, A., Pappas, G. D. &Bennett, M. V. L. (1972) Cobalt: A tracer for light and electron microscopy that can cross an electrotonic synapse.Journal of Cell Biology 55, 204a.
Politoff, A., Pappas, G. D. &Bennett, M. V. L. (1974) Cobalt ions cross an electrotonic synapse if cytoplasmic concentration is low.Brain Research 76, 343–6.
Rademakers.,L. H. P. M. (1977) Identification of a secretomotor centre in the brain ofLocusta migratoria, controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum.Cell and Tissue Research 184, 381–95.
Ribi, W. A. (1983) Electron microscopy of Golgi-impregnated neurons. InFunctional Neuroanatomy. Springer Series in Experimental Entomology (edited byStrausfeld, N. J.) Heidelberg, Berlin, New York: Springer Verlag. In press.
Schürmann, F.-W. (1980) Methods for special staining of synaptic sites. InNeuroanatomical Techniques. Insect Nervous System (edited byStrausfeld, N. J. &Miller, T. A.), pp. 241–61. New York, Heidelberg, Berlin: Springer Verlag.
Strausfeld, N. J. &Bassemir, U. K. (1983) Cobalt-coupled neurons of a giant fibre system in Diptera.Journal of Neurocytology 12, 971–91.
Strausfeld, N. J. &Obermayer, M. (1976) Resolution of intraneuronal and transsynaptic migration of cobalt in the insect visual and central nervous systems.Journal of Comparative Physiology 110, 1–12.
Székely, G. &Kosaras, B. (1976) Dendro-dendritic contact between frog motoneurons shown with the cobalt labelling technique.Brain Research 108, 194–8.
Székely, G. &Kosaras, B. (1977) Electron microscopic identification of postsynaptic dorsal root terminals: a possible substrate of dorsal root potentials in the frog spinal cord.Experimental Brain Research 29, 531–93.
Timm, F. (1958) Zur Histochemie des Ammonshorngebietes.Zeitschrift für Zellforschung und mikroskopische Anatomie 48, 548–55.
Tyrer, N. M. &Bell, E. M. (1974) The intensification of cobalt-filled neurone profiles using a modification of Timm's sulphide-silver method.Brain Research 73, 151–5.
Tyrer, N. M., Shaw, M. K. &Altman, J. S. (1980) Intensification of cobalt-filled neurons in sections (light and electron microscopy). InNeuroanatomical Techniques. Insect Nervous System (edited byStrausfeld, N. J. &Miller, T. A.), pp. 426–46. New York, Heidelberg, Berlin: Springer Verlag.
Venable, J. H. &Coggeshall, R. (1965) A simplified lead citrate stain for use in electron microscopy.Journal of Cell Biology 25, 407–8.
Weakly, J. N. (1973) The action of cobalt ions on neuromuscular transmission in the frog.Journal of Physiology 234, 597–612.
Wood, J. G., Wallace, R., Whitaker, J. &Cheung, W. Y. (1980) Immunocytochemical localization of calmodulin and a heat labile calmodulin-binding protein (C-M-BP80) in basal ganglia from mouse brain.Journal of Cell Biology 84, 66–76.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bassemir, U.K., Strausfeld, N.J. Cytology of cobalt-filled neurons in flies: cobalt deposits at presynaptic and postsynaptic sites, mitochondria and the cytoskeleton. J Neurocytol 12, 949–970 (1983). https://doi.org/10.1007/BF01153344
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01153344