May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Survival, Excitability and Transfection of Neurons in an Organotypic Culture of the Adult Zebrafish Retina
Author Affiliations & Notes
  • S. Kustermann
    Experimental Ophthalmology, University Eye Hospital, Tuebingen, Germany
  • S. Schmid
    Animal Physiology, Zoological Institute, University Tuebingen, Tuebingen, Germany
  • S. Bolz
    Experimental Ophthalmology, University Eye Hospital, Tuebingen, Germany
  • O. Biehlmaier
    Brain Research Institute, University of Zurich, Zurich, Switzerland
  • K.L. Kohler
    Experimental Ophthalmology, University Eye Hospital, Tuebingen, Germany
  • Footnotes
    Commercial Relationships  S. Kustermann, None; S. Schmid, None; S. Bolz, None; O. Biehlmaier, None; K.L. Kohler, None.
  • Footnotes
    Support  DFG Grant KO 1120/14
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1661. doi:
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      S. Kustermann, S. Schmid, S. Bolz, O. Biehlmaier, K.L. Kohler; Survival, Excitability and Transfection of Neurons in an Organotypic Culture of the Adult Zebrafish Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1661.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To survey cell survival, signal processing, and transfection techniques in long–time organotypic cultures of the adult retina. Methods: Isolated adult zebrafish retinas were cultivated over a period of 10 days in vitro (div). Morphological alterations were monitored consecutively by histological, immunocytological, and electron microscopic techniques. Electrophysiological recordings were carried out with the patch–clamp technique from cells in the ganglion cell layer (GCL). Plasmid DNA with an EGFP gene inserted was transferred into cells by electroporation in a boyden–chamber insert. Results: After 10 div the retinal cyto–architecture with its typical lamination was mainly unaltered and the inner parts of the retina were well preserved with all the major neuronal cell types present. However, borders between the nuclear and plexiform layers were somewhat obliterated due to the Muller glia that had become hypertrophic and reactive within the first 3div. TUNEL staining revealed only few apoptotic cells after 10div. Electron microscopy showed triade and diade synaptic structures in the photoreceptor terminals after 10div. Electrophysiological recordings from neurons in the GCL revealed that resting membrane potentials more negative than –50 mV were present over the entire in vitro period and some cells showed spontaneous activity. All cells were able to fire action potentials in response to a depolarizing current injection. Following electroporation of the cultivated retina we observed EGFP expression in neurons already after 12 hours. The expression remained stable over the whole culture period. Conclusions: Adult zebrafish retina can be kept and preserved in an organotypic culture up to 10 days. The culture system circumvents the limited accessibility of the retina in vivo and enables genetic as well as electrophysiological investigations.

Keywords: retinal culture 
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