Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina

Abstract

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.

Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1) and electroporation: methods and optogenetic applications

“…Electroporation has been used in zebrafish to introduce DNA constructs into larval neurons, adult retinal neurons or adult muscle cells (Rambabu et al, 2005; Hendricks and Jesuthasan, 2007; Bianco et al, 2008; Kustermann et al, 2008). However, the procedures used in these studies cannot be used to express transgenes in the adult zebrafish brain without major modifications.…”

Time course Analysis of Gene expression patterns in ZebrafIsh Eye during Optic Nerve Regeneration

“…In goldfish, studies show that 10%–20% of the RGCs are lost during a wave of cell death after ONX, which is a small number compared to mammals where nearly all RGCs undergo atrophy/apoptosis after injury 43–45. Another study using in vitro organotypic culture of adult zebrafish retina reported a rate of nearly 50% RGC apoptosis as determined by TUNEL staining of the retinal explants after 10 days in culture 46. This apoptosis has been attributed to the axotomy required to prepare the retinal explants 47.…”

“…This apoptosis has been attributed to the axotomy required to prepare the retinal explants 47. The loss of RGCs in the explants do not appear to be compensated by cell proliferation as determined by BrdU uptake and labeling, although extensive proliferation is observed among other cell types 46. These differences between in vivo and in vitro findings suggest that signals that are present in the intact organism, and lacking in the explanted retina, may be responsible for RGC survival and activation of axonal re-growth after ONX.…”

Organotypic culture of neural retina as a research model of neurodegeneration of ganglion cells

“…Most authors indicate damage to the axons forming the optic nerve to be the most likely cause of ganglion cell death. In all the described cases of optic nerve damage, symptoms of apoptotic death of ganglion cells (pyknosis, appearance of TUNEL positive staining) appeared no earlier than 5 to 7 days after the operation, which was shown both by morphological methods at the light and electron microscopic levels [22,23] and by TUNEL method [19]. In rats and mice that had experi enced optical nerve damage, all the ganglion layer cells were viable on days 1 3 [19,20], while in our case a signif icant portion of these cells (~41%) died two days after axo tomy.…”