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Synaptic Organization of an organotypic slice culture of the mammalian retina

Published online by Cambridge University Press:  02 June 2009

Marco Sassoè-Pognetto ,
Andreas Feigenspan ,
Joachim Bormann  and
Heinz Wässle
Marco Sassoè-Pognetto
Affiliation:
Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany Dipartimento di Anatomie e Fisiologia Umana, Università di Torino, Corso Massimo d'Azeglio 52, 1-10126 Torino, Italy
Andreas Feigenspan
Affiliation:
Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany
Joachim Bormann
Affiliation:
Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany
Heinz Wässle
Affiliation:
Max-Planck-Institut für Hirnforschung, Neuroanatomische Abteilung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany
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Abstract

Vertical Slices of postnatal day 6 (P6) rat retina were cut and cultured using the roller-tube technique. The organotypic differentiation during a culture period of up to 30 days has been described in a previous study (Feigenspan et al., 1993a). Here we concentrated on the synaptic organization in the retinal slice culture. Electron microscopy revealed the presence of ribbon synapses in the outer plexiform layer and conventional and ribbon syanpses in the inner plexiform layer. Immunofluroscence with antibodies that recognize specific subunits of GABAA or glycine receptors revealed a punctuate distribution of the receptors. They were aggregated in “hot spots” that correspond to a concentration of receptors at postsynaptic sites. Different isoforms of GABAA and glycine receptors occured in the slice cultures. The experiments show that there is a differentiation of synapses and a diversity of transmitter receptors in the slice cultures that is comparable to the in vivo retina.

Keywords

GABA receptorsGlycine receptorsGephyrinSlice cultureSynapses
Type
Research Articles
Information
Visual Neuroscience , Volume 13 , Issue 4 , July 1996 , pp. 759 - 771
Copyright
Copyright © Cambridge University Press 1996

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References

Barres, B.A., Silverstein, B.E., Corey, D.P. & Chun, L.L.Y. (1988). Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1, 791–803.CrossRefGoogle ScholarPubMed
Benke, D., Mertens, S., Trzeciak, A., Gillessen, D. & Möhler, H. (1991 a). Identification and immunohistochemical mapping of GABAA receptor subtypes containing the δ-subunit in rat brain. FEBS Letters 283, 145–149.CrossRefGoogle ScholarPubMed
Benke, D., Mertens, S., Trzeciak, A., Gillessen, D. & Möhler, H. (1991 b). GABAA receptors display association of γ2-subunit with α1- and β2/3-subunits. Journal of Biological Chemistry 266, 4478–4483.CrossRefGoogle Scholar
Boos, R., Schneider, H. & Wässle, H. (1993). Voltage- and transmittergated currents of All-amacrine cells in a slice preparation of the rat retina. Journal of Neuroscience 13, 2874–2888.CrossRefGoogle Scholar
Bormann, J. & Feigenspan, A. (1995). GABAC receptors. Trends in Neurosciences 18, 515–518.CrossRefGoogle ScholarPubMed
Caffé, A.R., Visser, H., Jansen, H.G. & Sanyal, S. (1989). Histotypic differentiation of neonatal mouse retina in organ culture. Current Eye Research 8, 1083–1092.CrossRefGoogle ScholarPubMed
Catsicas, S., Catsicas, M., Keyser, K.T., Karten, H.J., Wilson, M.C. & Milner, R.J. (1992). Differential expression of the presynaptic protein SNAP-25 in mammalian retina. Journal of Neuroscience Research 33, 1–9.CrossRefGoogle ScholarPubMed
Cohen, E.D., Zhou, Z.J. & Fain, G.L. (1994). Ligand-gated currents of alpha and beta ganglion cells in the cat retinal slice. Journal of Neurophysiology 72, 1260–1269.CrossRefGoogle ScholarPubMed
Craig, A.M., Blackstone, C.D., Huganir, R.L. & Banker, G. (1994). Selective clustering of glutamate and γ-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proceedings of the National Academy of Sciences of the U.S.A. 91, 12373–12377.CrossRefGoogle ScholarPubMed
Cutting, G.R., Lu, L., O'Hara, B.F., Kasch, L.M., Montrose-Rafizadeh, C., Donovan, D.M., Sdhimada, S., Antonarakis, S.E., Guggino, W.B., Uhl, G.R. & Kazazxian, H.H. (1991). Cloning of the γ-aminobutyric (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina. Proceedings of the National Academy of Sciences of the U.S.A. 88, 2673–2677.CrossRefGoogle Scholar
Cutting, G.R., Curristin, S., Zoghbi, H., O'Hara, B., Selding, M.F. & Uhl, G.R. (1992). Identification of a putative γ-aminobutyric acid (GABA) receptor subunit cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4. Genomics 12, 801–806.CrossRefGoogle ScholarPubMed
Dowling, J.E. & Boycott, B.B. (1966). Organization of the primate retina: electron microscopy. Proceedings of the Royal Society B (London) 166, 80–111.Google ScholarPubMed
Edwards, F.A., Konnerth, A., Sakmann, B. & Takahashi, T. (1989). A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflügers Archiv 414, 600–612.CrossRefGoogle ScholarPubMed
Enz, R., Brandstätter, J.H., Hartveit, E., Wässle, H. & Bormann, J. (1995). Expression of GABA receptor ρ1 and ρ2 subunits in the retina and brain of the rat. European Journal of Neuroscience 7, 1495–1501.CrossRefGoogle Scholar
Feigenspan, A. & Bormann, J. (1994 a). Differential pharmacology of GABAA and GABAC receptors on rat retinal bipolar cells. European Journal of Pharmacology, Molecular Pharmacology Section 288, 97–104.CrossRefGoogle ScholarPubMed
Feigenspan, A. & Bormann, J. (1994 b). Facilitation of GABAergic signaling in the retina by receptors stimulating adenylate cyclase. Proceedings of the National Academy of Sciences of the U.S.A. 91, 10893–10897.CrossRefGoogle ScholarPubMed
Feigenspan, A. & Bormann, J. (1994 c). Modulation of GABAC receptors in rat retinal bipolar cells by protein kinase C. Journal of Physiology 481, 325–330.CrossRefGoogle ScholarPubMed
Feigenspan, A., Bormann, J. & Wässle, H. (1993 a). Organotypic slice culture of the mammalian retina. Visual Neuroscience 10, 203–217.CrossRefGoogle ScholarPubMed
Feigenspan, A., Wässle, H., & Bormann, J. (1993 b). Pharmacology of GABA receptor Cl− channels in rat retinae bipolar cells. Nature 361, 159–162.CrossRefGoogle ScholarPubMed
Fisher, L. (1979). Development of synaptic arrays in the inner plexi-form layer of neonatal mouse retina. Journal of Comparative Neurology 187, 359–372.CrossRefGoogle Scholar
Froehner, S.C. (1993). Regulation of ion channel distribution at synapses. Annual Review of Neuroscience 16, 347–368.CrossRefGoogle ScholarPubMed
Gähwiler, B.H. (1981 a). Morphological differentiation of nerve cells in thin organotypic cultures derived from rat hippocampus and cerebellum. Proceedings of the Royal Society B (London) 211, 287–290.Google ScholarPubMed
Gähwiler, B.H. (1981 b). Organotypic monolayer cultures of nervous tissue. Journal of Neuroscience Methods 4, 329–342.CrossRefGoogle ScholarPubMed
Gähwiler, B.H. (1984). Development of the hippocampus in vitro: Cell types, synapses and receptors. Neuroscience 11, 751–760.CrossRefGoogle ScholarPubMed
Gähwiler, B.H., Thompson, S.M., Audinat, E. & Robertson, R.T. (1991). Organotypic slice cultures of neural tissue. In Culturing Nerve Cells, ed. Banker, G. & Kimberly, G., pp. 379–411. Cambridge, Massachusetts: MIT Press.Google Scholar
Gao, B., Fritschy, J.M., Benke, D. & Möhler, H. (1993). Neuron specific expression of GABAA receptor subtypes: Differential association of the α1- and α3-subunits with serotonergic and GABAergic neurons. Neuroscience 54, 881–892.CrossRefGoogle Scholar
Greferath, U., Brandstätter, J.H., Wässle, H., Kirsch, J., Kuhse, J. & Grünert, U. (1994). Differential expression of glycine receptor subunits in the retina of the rat: A study using immunocytochemistry and in situ hybridization. Visual Neuroscience 11, 721–729.CrossRefGoogle Scholar
Greferath, U., Grünert, U., Fritschy, J.M., Stephenson, A., Möhler, H. & Wässle, H. (1995). GABAA receptor subunits have differential distributions in the rat retina: In situ hybridization and immunohistochemistry. Journal of Comparative Neurology 353, 553–571.CrossRefGoogle ScholarPubMed
Grünert, U. & Wässle, H. (1993). Immunocytochemical localization of glycine receptors in the mammalian retina. Journal of Comparative Neurology 335, 523–537.CrossRefGoogle ScholarPubMed
Hartveit, E., Brandstätter, J.H., Sassoè-Pognetto, M., Laurie, D.J., Seeburg, P.H. & Wässle, H. (1994). Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina. Journal of Comparative Neurology 348, 570–582.CrossRefGoogle ScholarPubMed
Hild, W. & Callas, G. (1967). The behavior of retinal tissue in vitro, light and electron microscopic observations. Zeitschrift für Zellforschung 80, 1–21.CrossRefGoogle ScholarPubMed
Hollmann, M. & Heinemann, S. (1994). Cloned glutamate receptors. Annual Review of Neuroscience 17, 31–108.CrossRefGoogle ScholarPubMed
Jäger, J. & Wässle, H. (1987). Localization of glycine uptake and receptors in the cat retina. Neuroscience Letters 75, 147–151.CrossRefGoogle ScholarPubMed
Jahn, R. & Südhof, T.C. (1993). Synaptic vesicle traffic: Rush hour in the nerve terminal. Journal of Neurochemistry 61, 12–21.CrossRefGoogle ScholarPubMed
Kapfhammer, J.P., Christ, F. & Schwab, M.E. (1994). The expression of GAP-43 and synaptophysin in the developing rat retina. Developmental Brain Research 80, 251–260.CrossRefGoogle ScholarPubMed
Kirsch, J. & Betz, H. (1993). Widespread expression of gephyrin, a putative glycine receptor-tubulin linker protein, in rat brain. Brain Research 621, 301–310.CrossRefGoogle ScholarPubMed
Kirsch, J., Langosch, D., Prior, P., Littauer, U.Z., Schmitt, B. & Betz, H. (1991). The 93-kDa glycine receptor-associated protein binds to tubulin. Journal of Biological Chemistry 266, 22242–22245.CrossRefGoogle ScholarPubMed
Kirsch, J., Malosio, M.-L., Wolters, I. & Betz, H. (1993). Distribution of gephyrin transcripts in the adult and developing rat brain. European Journal of Neuroscience 5, 1109–1117CrossRefGoogle ScholarPubMed
Knöpfel, T., Vranesic, I., Gähwiler, B.H. & Brown, D.A. (1990). Muscarinic and β-adrenergic depression of the slow Ca2+-activated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of depolarization-induced cytosolic Ca2+ transients. Proceedings of the National Academy of Sciences of the U.S.A. 87, 4083–4087.CrossRefGoogle Scholar
Koulen, P., Sassoè-Pognetto, M., Grünert, U. & Wässle, H. (1996). Selective clustering of GABAA and glycine receptors in the mammalian retina. Journal of Neuroscience 16, 2127–2140.CrossRefGoogle ScholarPubMed
Kuhse, J., Betz, H. & Kirsch, J. (1995). The inhibitory glycine receptor—Architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex. Current Opinion in Neurobiology 5, 318–323.CrossRefGoogle ScholarPubMed
LaVail, M.M. & Hild, W. (1971). Histotypic organization of the rat retina in vitro. Zeitschrift für Zellforschung 114, 557–579.CrossRefGoogle Scholar
Llano, I., Marty, A., Johnson, J.W., Ascher, P. & Gähwiler, B.H. (1988). Patch-clamping of amino acid-activated responses in “organ-otypic” slice cultures. Proceedings of the National Academy of Sciences of the U.S.A. 85, 3221–3225.CrossRefGoogle Scholar
MacLeish, P.R. & Townes-Anderson, E. (1988). Growth and synapse formation among major classes of adult salamander retinal neurons in vitro. Neuron 1, 751–760.CrossRefGoogle ScholarPubMed
MacLeish, P.R., Barnstable, C.J. & Townes-Anderson, E. (1983). Use of a monoclonal antibody as a substrate for mature neurons in vitro. Proceedings of the National Academy of Sciences of the U.S.A. 80, 7014–7018.CrossRefGoogle ScholarPubMed
Mandell, J.W., Townes-Anderson, E., Czernik, A.J., Cameron, R., Greengard, P. & De Camilli, P. (1990). Synapsins in the vertebrate retina: Absence from ribbon synapses and heterogeneous distribution among conventional synapses. Neuron 5, 19–33.CrossRefGoogle ScholarPubMed
Marksitzer, R., Benke, D., Fritschy, J.M., Trzeciak, A., Bannwarth, W. & Möhler, H. (1993). GABAA receptors: Drug binding profile and distribution of receptors containing the α2-subunit in situ. Journal of Receptor Research 13, 467–477.CrossRefGoogle Scholar
Maslim, J. & Stone, J. (1986). Synaptogenesis in the retina of the cat. Brain Research 373, 35–48.CrossRefGoogle ScholarPubMed
Maslim, J. & Stone, J. (1985). Early differentiation of ganglion cells in the cat's retina. Proceedings of the Anatomy Society of Australia (New Zealand) 3 P.Google Scholar
McMahan, U.J. (1990). The agrin hypothesis. Cold Spring Harbor Symposia on Quantitative Biology 55, 407–418.CrossRefGoogle ScholarPubMed
Missotten, L. (1965). The synapses in the human retina. In The Structure of the Eye. II. Symposium, ed. Rohen, J.W., pp. 17–28. Stuttgart: Schattauer.Google Scholar
Nishimura, Y. & Rakic, P. (1987). Development of the Rhesus monkey retina: II. A three-dimensional analysis of the sequences of synaptic combinations in the inner plexiform layer. Journal of Comparative Neurology 262, 290–313.CrossRefGoogle Scholar
Nomura, A., Shigemoto, R., Nakamura, Y., Okamoto, N., Mizuno, N. & Nakanishi, S. (1994). Developmentally regulated postsynaptic localization of a metabotropic glutamate receptor in rat rod bipolar cells. Cell 77, 361–369.CrossRefGoogle ScholarPubMed
Nusser, Z., Roberts, J.D.B., Baude, A., Richards, J.G. & Somogyi, P. (1995). Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method. Journal of Neuroscience 15, 2948–2960.CrossRefGoogle ScholarPubMed
Pfeiffer, F., Simler, R., Grenninoloh, G. & Betz, H. (1984). Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proceedings of the National Academy of Sciences of the U.S.A. 81, 7224–7227.CrossRefGoogle ScholarPubMed
Polenzani, L., Woodward, R.M. & Miledi, R. (1991). Expression of mammalian γ-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proceedings of the National Academy of Sciences of the U.S.A. 88, 4318–4322.CrossRefGoogle ScholarPubMed
Pourcho, R.G. & Owczarzak, M.T. (1991 a). Connectivity of glycine immunoreactive amacrine cells in the cat retina. Journal of Comparative Neurology 307, 549–561.CrossRefGoogle ScholarPubMed
Pourcho, R.G. & Owczarzak, M.T. (1991 b). Glycine receptor immunoreactivity is localized at amacrine synapses in cat retina. Visual Neuroscience 7, 611–618.CrossRefGoogle ScholarPubMed
Pow, D.V. & Crook, D.K. (1994). Rapid postmortem changes in the cellular localisation of amino acid transmitters in the retina as assessed by immunocytochemistry. Brain Research 653, 199–209.CrossRefGoogle ScholarPubMed
Pow, D.V., Wright, L.L. & Vaney, D.I. (1995). The immunocytochemical detection of amino-acid neurotransmitters in paraformaldehyde-fixed tissues. Journal of Neuroscience Methods 56, 115–123.CrossRefGoogle ScholarPubMed
Rickman, D.W. (1995). BDNF enhances differentiation of neurons of the rod pathway in organotypic cultures of neonatal rat retina. Society for Neuroscience Abstracts 21, 611.15.Google Scholar
Robinson, S.R. (1991). Development of the mammalian retina. In Neuroanatomy of the Visual Pathwaysand their Development, Vision and Visual Dysfunction (Series), Vol. 3, ed. Dreher, B. & Robinson, S.R., pp. 69–128. UK:Macmillan.Google Scholar
Sarthy, P.V. & Bacon, W. (1985). Developmental expression of a synaptic vesicle-specific protein in the rat retina. Developmental Biology 112, 284–291.CrossRefGoogle ScholarPubMed
Sassoè-Pognetto, M., Wässle, H. & Grünert, U. (1994). Glycinergic synapses in the rod pathway of the rat retina: Cone bipolar cells express the al subunit of the glycine receptor. Journal of Neuroscience 14, 5131–5146.CrossRefGoogle Scholar
Sassoè-Pognetto, M., Kirsch, J., Grünert, U., Greferath, U., Fritschy, J.M., Möhler, H., Betz, H. & Wässle, H. (1995). Colocalization of gephyrin and GABAA receptor subunits in the rat retina. Journal of Comparative Neurology 357, 1–14.CrossRefGoogle ScholarPubMed
Schmitt, B., Knaus, P., Becker, C.M. & Betz, H. (1987). The Mr 93,000 polypeptide of the postsynaptic glycine receptor complex is a peripheral membrane protein. Biochemistry 26, 805–811.CrossRefGoogle ScholarPubMed
Schröder, S., Hoch, W., Becker, C.-M., Grenningloh, G. & Betz, H. (1991). Mapping of antigenic epitopes of the al subunit of the inhibitory glycine receptor. Biochemistry 30, 4247.Google Scholar
Sieghart, W. (1995). Structure and pharmacology of γ-aminobutyric acidA receptor subtypes. Pharmacological Reviews 47, 181–234.Google ScholarPubMed
Sparrow, J.R., Hicks, D. & Barnstable, C.J. (1990). Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina. Developmental Brain Research 51, 69–84.CrossRefGoogle ScholarPubMed
Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, F.-M.S. & Ottersen, O.P. (1983). First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517–520.CrossRefGoogle ScholarPubMed
Townes-Anderson, E., MacLeish, P.R. & Raviola, E. (1985). Rod cells dissociated from mature salamander retina: Ultrastructure and uptake of horseradish peroxidase. Journal of Cell Biology 100, 175–188.CrossRefGoogle ScholarPubMed
Triller, A., Cluzeaud, F., Pfeiffer, F., Betz, H. & Korn, H. (1985). Distribution of glycine receptors at central synapses: An immunoelectron microscopy study. Journal of Cell Biology 101, 683–688.CrossRefGoogle ScholarPubMed
Ullrich, B. & Südhof, T.C. (1994). Distribution of synaptic markers in the retina: Implications for synaptic vesicle traffic in ribbon synapses. Journal of Physiology 88, 249–257.Google ScholarPubMed
Vaney, D.I. (1990). The mosaic of amacrine cells in the mammalian retina. Progress in Retinal Research 9, 49–100.CrossRefGoogle Scholar
Vardi, N. & Auerbach, P. (1995). Specific cell types in cat retina express different forms of glutamic acid decarboxylase. Journal of Comparative Neurology 351, 374–384.CrossRefGoogle ScholarPubMed
Wässle, H. & Chun, M.H. (1989). GABA-like immunoreactivity in the cat retina: Light microscopy. Journal of Comparative Neurology 279, 43–54.CrossRefGoogle ScholarPubMed
Weidman, T.A. & Kuwabara, T. (1968). Postnatal development of the rat retina. An electron microscopy study. Archives of Ophthalmology 79, 470–484.CrossRefGoogle Scholar
Woodward, R.M., Polenzani, L. & Miledi, R. (1992). Characterization of bicuculline/baclofen-insensitive γ-aminobutyric acid receptors expressed in Xenopus oocytes. I. Effect of Cl− channel inhibitors. Molecular Pharmacology 42, 165–173.Google ScholarPubMed
Woodward, R.M., Polenzani, L. & Miledi, R. (1993). Characterization of bicuculline/baclofen-insensitive (ρ-like) γ-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of γ-aminobutyric acidA and γ-aminobutyric acidB receptor agonists and antagonists. Molecular Pharmacology 43, 609–625.Google ScholarPubMed
Yazulla, S. & Studholme, K.M. (1991 a). Glycinergic interplexiform cells make synaptic contact with amacrine cell bodies in goldfish retina. Journal of Comparative Neurology 310, 1–10.CrossRefGoogle ScholarPubMed
Yazulla, S. & Studholme, K.M. (1991 b). Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses. Journal of Comparative Neurology 310, 11–20.CrossRefGoogle ScholarPubMed
Zhou, Z.J., Marshak, D.W. & Fain, G.L. (1994). Amino acid receptors of midget and parasol ganglion cells in primate retina. Proceedings of the National Academy of Sciences of the U.S.A. 91, 4907–4911.CrossRefGoogle ScholarPubMed