Journal of Neurocytology

, Volume 17, Issue 6, pp 859–870 | Cite as

Localization of GABA and glycine in goldfish retina by electron microscopic postembedding immunocytochemistry: improved visualization of synaptic structures with LR White resin

  • Keith M. Studholme
  • Stephen Yazulla


A post-embedding, electron microscopic immunocytochemistry technique, modified from existing protocols, was used to examine the labelling patterns of GABA immunoreactivity and glycine immunoreactivity in goldfish retina. Retinae were fixed in mixed aldehyde solution, dehydrated in ethanol, staineden bloc with uranyl acetate and phosphotungstic acid and embedded in LR White resin. Substances were localized in thin sections by floating grids first on a drop of primary antiserum and then on a colloidal gold-IgG conjugate. Finally, grids were exposed to osmium vapour. The localization of GABA immunoreactivity matched that of [3H]-GABA uptake or glutamate decarboxylase immunoreactivity as described previously. In the outer retina, GABA immunoreactivity was found in the cell bodies and axon terminals of H1 horizontal cells and their dendrites opposite cone photoreceptor terminals. Selected amacrine cell bodies were labelled, as were many processes, both synaptic and non-synaptic, throughout the inner plexiform layer, including most amacrine cell processes contacting the synaptic terminals of type Mb bipolar cells. Numerous amacrine cells, their processes in the inner and outer plexiform layers, and photoreceptor terminals contained glycine immunoreactivity in a distribution similar to that shown by [3H]-glycine uptake. Despite the absence of osmium in the primary or secondary fixative, our protocol results in excellent visibility of synaptic structures and detectability of the colloidal gold immunolabel. Also, it does not cause extraction of the HRP/DAB reaction product and is therefore suitable for double-label analysis of neurons labelled with horseradish peroxidase.


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  1. Bachmann, S., Gilbert, P. &Minuth, W. W. (1987) Electron-microscopic immunogold localization of a collecting-duct antigen (PCD2) in the intercalated and principal cells of the rabbit kidney.Cell and Tissue Research 249, 633–40.Google Scholar
  2. Ball, A. K. (1987) Immunocytochemical and autoradiographic localization of GABAergic neurons in the goldfish retina.Journal of Comparative Neurology 255, 317–25.Google Scholar
  3. Ball, A. K. &Brandon, C. (1986) Localization of [3H]-GABA, -muscimol and -glycine in goldfish retinas stained for glutamate decarboxylase.Journal of Neuroscience 6, 1621–7.Google Scholar
  4. Bendayan, M., Nanci, A. &Kan, F. W. K. (1987) Effect of tissue processing on colloidal gold cytochemistry.Journal of Histochemistry and Cytochemistry 35, 983–96.Google Scholar
  5. Bendayan, M. &Zollinger, M. (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.Journal of Histochemistry and Cytochemistry 31, 101–9.Google Scholar
  6. Bird, A. C., Farber, D. B., Kreiger, A. E., Straatsma, B. R. &Bok, D. (1988a) The potential usefulness to research of retina obtained by biopsy.Investigative Ophthalmology and Visual Science 29, 2–11.Google Scholar
  7. Bird, A. C., Flannery, J. G. &Bok, D. (1988b) A diurnal rhythm in opsin content ofRana pipien rod inner segments.Investigative Ophthalmology and Visual Science 29, 1028–39.Google Scholar
  8. Bloom, F. E. &Aghajanian, G. K. (1966) Cytochemistry of synapses: selective staining for electron microscopy.Science 15, 1575–7.Google Scholar
  9. Bok, D., Ong, D. E. &Chytil, F. (1984) Immunocytochemical localization of cellular retinol binding protein in the rat retina.Investigative Ophthalmology and Visual Science 25, 877–83.Google Scholar
  10. Cajal, S. R. Y. (1933)The Structure of the Retina (compiled and translated byThorpe, S. A. &Glickstein, M.), Springfield: Charles C. Thomas (1972).Google Scholar
  11. Causton, B. E. (1984) The choice of resins for electron immunocytochemistry. InImmunolabelling for Electron Microscopy (edited byPolar, J. M. &Varndell, I. M.), pp. 29–36. The Netherlands: Elsevier Science Publishers.Google Scholar
  12. Chaitin, M. H. &Bok, D. (1986) Immunoferritin localization of actin in retinal photoreceptors.Investigative Ophthalmology and Visual Science 27, 1764–7.Google Scholar
  13. Eldred, W. D., Zucker, C., Karten, H. J. &Yazulla, S. (1983) Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry.Journal of Histochemistry and Cytochemistry 31, 285–92.Google Scholar
  14. Erickson, P. A., Anderson, D. H. &Fisher, S. K. (1987) Use of uranyl acetate en bloc to improve tissue preservation and labeling for post embedding immunoelectron microscopy.Journal of Electron Microscopy Technique 5, 303–14.Google Scholar
  15. Gräber, M. B. &Kreutzberg, G. W. (1985) Immuno gold staining (IGS) for electron microscopical demonstration of glial fibrillary acidic (GFA) protein in LR White embedded tissue.Histochemistry 83, 497–500.Google Scholar
  16. Lam, D. M. K. &Steinman, L. (1971) The uptake of [3H]-γ-aminobutyric acid in the goldfish retina.Proceedings of the National Academy of Sciences USA 68, 2777–81.Google Scholar
  17. Marc, R. E. (1985) The role of glycine in retinal circuitry. InRetinal Transmitters and Modulators: Models for the Brain, Vol. 1 (edited byMorgan, W. W.), pp. 119–58. Boca Raton, Florida: CRC Press.Google Scholar
  18. Marc, R. E. &Lam, D. M. K. (1981) Glycinergic pathways in goldfish retina.Journal of Neuroscience 1, 152–65.Google Scholar
  19. Marc, R. E., Stell, W. K., Bok, D. &Lam, D. M. K. (1978) GABAergic pathways in the goldfish retina.Journal of Comparative Neurology 182, 221–46.Google Scholar
  20. Mosinger, J. L., Studholme, K. &Yazulla, S. (1986) Immunocytochemical localization of GABA in the retina: A species comparison.Experimental Eye, Research 42, 631–44.Google Scholar
  21. Newman, G. R. &Hobot, J. A. (1987) Modern acrylics for post-embedding immunostaining techniques.Journal of Histochemistry and Cytochemistry 35, 971–81.Google Scholar
  22. Newman, G. R., Jasani, B. &Williams, E. D. (1983) A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope.Histochemical Journal 15, 543–55.Google Scholar
  23. Ring, P. K. M. &Johanson, V. (1987) Immunoelectron microscopic demonstration of thyroglobulin and thyroid hormones in rat thyroid gland.Journal of Histochemistry and Cytochemistry 35, 1095–104.Google Scholar
  24. Roth, J., Bendayan, M., Carlemalm, E., Villiger, W. &Garavito, M. (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.Journal of Histochemistry and Cytochemistry 29, 663–71.Google Scholar
  25. Somogyi, P. &Hodgson, A. J. (1985) Antiserum to gamma amino butyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of the cat striate cortex.Journal of Histochemistry and Cytochemistry 33, 249–57.Google Scholar
  26. Somogyi, P. &Soltesz, I. (1986) Immunogold demonstration of GABA in synaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat's visual cortex.Neuroscience 19, 1051–65.Google Scholar
  27. Stell, W. K. (1967) The structure and relationships of horizontal cells and photoreceptor-bipolar synaptic complexes in goldfish retina.American Journal of Anatomy 120, 401–24.Google Scholar
  28. Stell, W. K. (1975) Horizontal cell axons and axon terminals in goldfish retina,Journal of Comparative Neurology 159, 503–20.Google Scholar
  29. Sternberger, L. A. (1979)Immunocytochemistry (2nd edn). New York: John Wiley and Sons.Google Scholar
  30. Studholme, K. M. &Yazulla, S. (1988) Localization of glycine-immunoreactivity in the goldfish retina.Investigative Ophthalmology and Visual Science, Suppl.29, 273 (Abstract).Google Scholar
  31. Tachibana, M. &Kaneko, A. (1987) γ-aminobutyric acid exerts a local inhibitory action on the axon terminal of bipolar cells: evidence for negative feedback from amacrine cells.Proceedings of the National Academy of Sciences USA 84, 3501–5.Google Scholar
  32. Timms, B. G. (1986) Post embedding immunogold labeling for electron microscopy using “LR White” resin.American Journal of Anatomy 175, 267–75.Google Scholar
  33. Van Den Pol, A. N. (1985) Dual ultrastructural localization of two neurotransmitter-related antigens: colloidal goldlabeled neurophysin-immunoreactive supraoptic neurons receive peroxidase-labeled glutamate decarboxylase- or gold-labeled GABA-immunoreactive synapses,Journal of Neuroscience 5, 2940–54.Google Scholar
  34. Wenthold, R. (1987) Evidence for a glycinergic pathway connecting the two cochlear nuclei: an immunocytochemical and retrograde transport study,Brain Research 415, 183–7.Google Scholar
  35. Wenthold, R., Zemple, J., Parakkal, M. A. &Altschuler, R. (1986) Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig.Brain Research 380, 7–18.Google Scholar
  36. Yang, C.-Y. &Yazulla, S. (1988) Double-label EM visualization of transmitter input to physiologically identified ganglion cells in tiger salamander retina.Investigative Ophthalmology and Visual Science, Suppl.29, 197 (Abstract).Google Scholar
  37. Yazulla, S. (1986) GABAergic mechanisms in the retina. InProgress in Retinal Research, Vol. 5 (edited byOsborne, N. N. &Chader, C. J.), pp. 1–52. Oxford: Pergamon Press.Google Scholar
  38. Yazulla, S., Studholme, K. M. &Wu, J.-Y. (1986) Comparative distribution of [3H]-GABA uptake and GAD-immunocytochemistry in goldfish retinal amacrine cells: a double-label analysis.Journal of Comparative Neurology 244, 149–62.Google Scholar
  39. Yazulla, S., Studholme, K. &Wu, J.-Y. (1987) GABAergic input to the synaptic terminals of Mb1 bipolar cells in the goldfish retina.Brain Research 411, 400–5.Google Scholar
  40. Zucker, C., Yazulla, S. &Wu, J.-Y. (1984) Non-correspondence of [3H]-GABA uptake and GAD-localization in goldfish amacrine cells.Brain Research 298, 154–8.Google Scholar

Copyright information

© Chapman and Hall Ltd 1988

Authors and Affiliations

  • Keith M. Studholme
    • 1
  • Stephen Yazulla
    • 1
  1. 1.Department of Neurobiology and BehaviorState University of New YorkStony BrookUSA

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