Summary
Retinae from two- and three-day-old rats were explanted in plasma clots and grown in vitro with the flying coverslip method. After seven to seventeen days in culture, the retinal tissue was fixed with aldehydes and osmium tetroxide and embedded for examination with the electron microscope. Study of cross sections (perpendicular to the coverslip) revealed a histotypic pattern of organization, especially in the thicker regions of the explants. Layering of cells quite similar to that in the intact retina was seen to develop from the relatively primitive, explanted retinal epithelium. However, each layer contained fewer cells than its counterpart in vivo. All major neuronal cell types were distinguished by their location and cytological characteristics. Development of the saccules of sensory cell outer segments was observed to occur in vitro by an infolding of the plasma membrane. Synaptic ribbon complexes developed to the mature form in the outer plexiform layers, while conventional synapses were numerous in the inner plexiform layers. Synaptic ribbons were also seen in bipolar cell axons in the inner plexiform layers. Amacrine and ganglion cells in these regions were relatively sparse. A survey of posterior regions of noncultured three-day-old rat retinae showed no synapses of any sort; therefore the synapses in the cultures formed in vitro. The retina is recommended for studies of synaptogenesis in tissue culture, for it offers several advantages over expiants from other areas of the neuraxis, including a clear layering pattern, many identifiable cell processes with characteristic synaptic relationships between them, and a well-defined sequence of developmental events.
Zusammenfassung
Netzhäute von 2–3 Tage alten Ratten wurden in Plasma auf Deckgläsern in Rollerröhrchen zur Kultur angesetzt. Nach 7–17 Tagen in vitro wurden die Kulturen mit Aldehyden und Osmiumsäure fixiert und für elektronenmikroskopische Untersuchung weiterverarbeitet. Gewebsquerschnitte (senkrecht zum Deckglas) zeigten histotypische Organisation, besonders in den dickeren Abschnitten der Explantate. Die Schichtung der Zellen entwickelte sich ganz ähnlich derjenigen in der Retina in situ aus dem relativ primitiven ausgepflanzten Netzhautepithel, jedoch enthielten die verschiedenen Schichten weniger Zellen als in der Retina in vivo. Alle Hauptnervenzelltypen konnten auf Grund ihrer Lokalisation und ihrer cytologischen Merkmale unterschieden werden. Die Entstehung von membranösen Lamellen in den Außengliedern der Sinneszellen konnte als Einfaltung der Plasmamembran beobachtet werden. Synaptische Bandkomplexe in ausgereifter Form wurden in der äußeren plexiformen Schicht nachgewiesen, während konventionelle Synapsen in der inneren plexiformen Schicht häufig angetroffen wurden. Synaptische Bänder waren ebenfalls in den Axonen bipolarer Zellen in der inneren plexiformen Schicht nachweisbar. Amakrine und Ganglienzellen waren in diesen Regionen ziemlich selten vertreten. Da die Untersuchung von nicht kultivierten Netzhäuten drei Tage alter Tiere keinerlei Synapsen zeigte, wird geschlossen, daß die Synapsen in den Kulturen in vitro entstanden sein müssen. Die Netzhaut stellt ein günstiges Modell für die Synaptogenese in vitro dar, indem sie verschiedene Vorzüge vor Explantaten aus anderen Regionen des Zentralnervensystems aufweist, nämlich eine klare Schichtung, zahlreiche identifizierbare Zellfortsätze mit charakteristischen synaptischen Beziehungen und eine wohl definierte Folge von Entwicklungsvorgängen.
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References
Anderson, W. A., Ellis, R. A.: Ultrastructure of Trypanosoma lewisi: flagellum, microtubules, and the kinetoplast. J. Protozool. 12, 483–499 (1965).
Bok, D.: An electron microscopic analysis of migration, division and differentiation of presumptive rat photoreceptors. Anat. Rec. 160, 319 (Abstract) (1968).
Bornstein, M. B.: Observations on mouse nerve tissues in culture. NINDB monograph No. 2, Slow, latent, and temperature virus infections, pp. 177–185 (1965).
Bunge, M. B., Bunge, R. P., Peterson, E. R.: The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Brain Res. 6, 728–749 (1967).
Bunge, R. P., Bunge, M. B., Peterson, E. R.: An electron microscope study of cultured rat spinal cord. J. Cell Biol. 24, 163–191 (1965).
Callas, G.: Personal communication (1967).
—, Hild, W.: Electron microscopic observations of synaptic endings in cultures of mammalian central nervous tissue. Z. Zellforsch. 63, 686–691 (1964).
Cohen, A. I.: Vertebrate retinal cells and their organization. Biol. Rev. 38, 427–459 (1963).
Detwiler, S. R.: Experimental observations upon the developing rat retina. J. comp. Neurol. 55, 473–492 (1932).
Dowling, J. E.: The organization of vertebrate visual receptors. In: Molecular organization and biological function, J. M. Allen, ed., chapt. 7, p. 186–210. New York: Harper and Row 1967.
—: Synaptic organization of the frog retina: an electron microscopic analysis comparing the retinas of frogs and primates. Proc. roy. Soc. B. 170, 205–228 (1968).
-Dowling, J. E., Boycott, B. B.: Neural connections of the retina: Fine structure of the inner plexiform layer. Cold Spr. Harb. Symp. quant. Biol. 30, 393–402 (1965).
—, Brown, J. E., Major, D.: Synapses of horizontal cells in rabbit and cat retinas. Science 153, 1639–1641 (1966).
—, Gibbons, I. R.: The effect of vitamin A deficiency on the fine structure of the retina. In: The structure of the eye, G. K. Smelser, eed., p. 85–99, New York: Academic Press 1961.
—: The fine structure of the pigment epithelium in the albino rat. J. Cell Biol. 14, 459–474 (1962).
Droz, B.: Dynamic condition of proteins in the visual cells of rats and mice as shown by radioautography with labeled amino acids. Anat. Rec. 145, 157–168 (1963).
Duncan, D., Hild, W.: Mitochondrial alterations in cultures of the central nervous system as observed with the electron microscope. Z. Zellforsch. 51, 123–135 (1960).
Fine, B. S.: Limiting membranes of the sensory retina and pigment epithelium. Arch. Ophthal. 66, 847–860 (1961).
—: Synaptic lamellas in the human retina: an electron microscopic study. J. Neuropath. exp. Neurol. 22, 255–262 (1962).
Govardovsky, V. L., Kharkeevitch, T. A.: Histochemical and electron microscpical investigation on the development of photoreceptive cells under the conditions of tissue culture. Arkh. Anat. Gistol. Embriol. 49, 50–55 (1965) [Russisch].
Grainger, F., James, D. W., Tresman, R. L.: An electron-microscopic study of the early outgrowth from chick spinal cord in vitro. Z. Zellforsch. 90, 53–67 (1968).
Guillery, R. W., Sobkowicz, H. M., Scott, G. L.: Light and electron microscopical observations of the ventral horn and ventral root in long term cultures of the spinal cord of the fetal mouse. J. comp. Neurol. 134, 433–476 (1968).
Hansson, H.-A., Sourander, P.: Studies on cultures of mammalian retina. Z. Zellforsch. 62, 26–47 (1964).
Hild, W.: Myelogenesis in cultures of mammalian central nervous tissue. Z. Zellforsch. 46, 71–95 (1957a).
—: Ependymal cells in tissue culture. Z. Zellforsch. 46, 259–271 (1957b).
—: Cell types and neuronal connections in cultures of mammalian central nervous tissue. Z. Zellforsch. 69, 155–188 (1966).
—, Callas, G.: The behavior of retinal tissue in vitro, light and electron microscopic observations. Z. Zellforsch. 80, 1–21 (1967).
—, Tasaki, I.: Morphological and physiological properties of neurons and glial cells in tissue culture. J. Neurophysiol. 25, 277–304 (1962).
James, D. W., Tresman, R. L.: Synaptic profiles in the outgrowth from chick spinal cord in vitro. Z. Zellforsch. 101, 598–606 (1969).
Karnovsky, M. J.: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137–138 (Abstract) 1965.
Kolb, H.: Organization of the outer plexiform layer of the primate retina: Electron microscopy of Golgi-impregnated cells. Phil. Trans. B 258, 261–283 (1970).
Kroll, A. J., Machemer, R.: Experimental retinal detachment in the owl monkey. III. Electron microscopy of retina and pigment epithelium. Amer. J. Ophthal. 66, 410–427 (1968).
Ladman, A. J.: The fine structure of the rod-bipolar cell synapse in the retina of the albino rat. J. biophysic. biochem. Cytol. 4, 459–466 (1958).
LaVail, M. M.: A method of embedding selected areas of tissue cultures for electron microscopy. Tex. Rep. Biol. Med. 26, 215–222 (1968).
- Formation of extracellular nuclear masses in developing cultures of rat retina. In preparation (1971).
Liss, L., Wolter, J. R.: Human retinal neurons in tissue culture. Amer. J. Ophthal. 52, 834–841 (1961).
Lucas, D. R.: In vitro maintenance of the mature guinea-pig retina. Vision Res. 2, 35–41 (1962).
—: Special cytology of the eye. In: Cells and tissues in culture, E. N. Willmer, ed., vol. 2, p. 457–520. New York: Academic Press 1965.
—, Trowell, O. A.: In vitro culture of the eye and the retina of the mouse and rat. J. Embryol. exp. Morph. 6, 178–182 (1958).
Masurovsky, E. B., Bunge, R. P.: Fluoroplastic coverslips for long-term nerve tissue culture. Stain Technol. 43, 161–165 (1968).
Meller, K., Eschner, J.: Vergleichende Untersuchungen über die Feinstruktur der Bipolarzellschicht der Vertebratenretina. Z. Zellforsch. 68, 550–567 (1965).
—, Haupt, R.: Die Feinstruktur der Neuro-, Glio-, und Ependymoblasten von Hühnerembryonen in der Gewebekultur. Z. Zellforsch. 76, 260–277 (1967).
Missotten, L.: The synapses in the human retina. In: The structure of the eye. II. Symposium, J. W. Rohen, ed., p. 17–28. Stuttgart: Schattauer 1965.
Murray, M. R.: Nervous tissues in vitro. In: Cells and tissues in culture. Methods, biology, and physiology, E. N. Willmer, ed., vol. 2, p. 373–455. New York: Academic Press 1965.
Nilsson, S. E. G.: Receptor cell outer segment development and ultrastructure of the disk membranes in the retina of the tadople (Rana pipiens). J. Ultrastruct. Res. 11, 581–620 (1964).
Olney, J. W.: Centripetal sequence of appearance of receptor-bipolar synaptic structures in developing mouse retina. Nature (Lond.) 218, 281–282 (1968a).
—: An electron microscopic study of synapse formation, receptor outer segment development, and other aspects of developing mouse retina. Invest. Ophthal. 7, 250–268 (1968b).
Pei, Y. F., Smelser, G. K.: Some fine structural features of the ora serrata region in primate eyes. Invest. Ophthal. 7, 672–688 (1968).
Pellegrino de Iraldi, A., Jaim Etcheverry, G.: Granulated vesicles in retinal synapses and neurons. Z. Zellforsch. 81, 283–296 (1967).
Peterson, E. R., Crain, S. M., Murray, M. R.: Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human). Z. Zellforsch. 66, 130–154 (1965).
Pomerat, C. M., Costero, I.: Tissue cultures of cat cerebellum. Amer. J. Anat. 99, 211–222 (1956).
Raviola, G., Raviola, E.: Light and electron microscopic observations on the inner plexiform layer of the rabbit retina. Amer. J. Anat. 120, 403–426 (1967).
Reynolds, E. S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963).
Richardson, T. M.: Cytoplasmic and ciliary connections between the inner and outer segments of mammalian visual receptors. Vision Res. 9, 727–731 (1969).
Sheffield, J. B.: Microtubules in the outer nuclear layer of rabbit retina. J. Microsc. 5, 173–180 (1966).
Sidman, R. L.: Tissue culture studies of inherited retinal dystrophy. Dis. nerv. Syst. 22, (4), 14–20 (1961).
Sobkowicz, H. M., Guillery, R. N., Bornstein, M. B.: Neuronal organization in long term cultures of the spinal cord of the fetal mouse. J. comp. Neurol. 132, 365–396 (1968).
Stefanelli, A., Zacchei, A. M., Caravita, S., Cataldi, E., Ieradi, L. A.: Sinapsi in vitro da cellule disgregate di retina di embrione di pollo. R. C.Acead. naz. Lincei 40, 758–762 (1966a).
Stefanelli, A. et al.: Differenziamento in riaggregati di retina in vitro. Arch. Zool. ital. 51, 985–996 (1966b).
—: Differenziamento di fotorecettori di pollo in riaggregati coltivati in vitro. R. C. Accad. naz. Lincei 42, 594–599 (1967a).
—: New-forming retinal synapses in vitro. Experientia (Basel) 23, 1–5 (1967b).
Stell, W. K.: Correlation of retinal cytoarchitecture and ultrastructure in Golgi preparations. Anat. Rec. 153, 389–398 (1965).
—: The structure and relationships of horizontal cells and photoreceptor-bipolar synaptic complexes in goldfish retina. Amer. J. Anat. 121, 401–424 (1967).
Tansley, K.: The formation of rosettes in the rat retina. Brit. J. Ophthal. 17, 321–336 (1933).
Tokuyasu, K., Yamada, E.: The fine structure of the retina. V. Abnormal retinal rods and their morphogenesis. J. biophys. biochem. Cytol. 7, 187–190 (1960).
Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475–478 (1958).
Weidman, T. A., Kuwabara, T.: Postnatal development of the rat retina. Arch. Ophthal. 79, 470–484 (1968).
Wolf, M. K.: Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum. J. Cell Biol. 22, 259–280 (1964).
—: Anatomy of cultured mouse cerebellum. II; Organotypic migration of granule cells demonstrated by silver impregnation of normal and mutant cultures. J. comp. Neurol. 140, 281–298 (1970).
—, Dubois-Dalcq, M.: Anatomy of cultured mouse cerebellum. I. Golgi and electron microscopic demonstration of granule cells, their afferent and efferent synapses. J. comp. Neurol. 140, 261–280 (1970).
Yamada, E., Ishikawa, T.: Some observations on the submicroscopic morphogenesis of the human retina. In: The structure of the eye, II. Symposium, J. W. Rohen, ed., p. 5–16. Stuttgart: Schattauer 1965.
Young, R. W.: Renewal of photoreceptor outer segments. Anat. Rec. 151, 484 (Abstract) (1965).
—: Passage of newly formed protein through the connecting cilium of retinal rods in the frog. J. Ultrastruct. Res. 23, 462–473 (1968).
—, Droz, B.: The renewal or protein in retinal rods and cones. J. Cell Biol. 39, 169–184 (1968).
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Dedicated to Professor Wolfgang Bargmann on the occasion of his 65th birthday.
This work was supported by USPHS Grants 5R01 NB 03114, 5T01 GM00459, and 5R01 NB 00690 from the National Institutes of Health, Bethesda, Maryland.
Sincere appreciation is expressed to Mrs. Eleanor Morris for her assistance in the management of the cultures, and to Mr. E. E. Pitsinger, Jr. for his photographic assistance.
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LaVail, M.M., Hild, W. Histotypic organization of the rat retina in vitro. Z. Zellforsch. 114, 557–579 (1971). https://doi.org/10.1007/BF00325640
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DOI: https://doi.org/10.1007/BF00325640