Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina
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Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro.
KeywordsNeurogenesis Adult retina Regeneration In vitro system Visual system Zebrafish, Danio rerio (Teleostet)
We thank S. Conrad (University of Tübingen, Germany) for technical support with the electroporation procedure and S. Bolz for excellent technical assistance with the immunhistochemistry. We are also grateful to K. W. Koch (University of Oldenburg, Germany) and J. Scholes (University College London, UK) for providing recoverin and 74C4 antibodies, respectively. Many thanks are also due to Theo van Veen (University of Tübingen, Germany) for helpful comments on the manuscript.