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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)
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.
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