Figure 2

Figure 2

Long-term primary cell culture of adult zebrafish retinal neurons at DIV 1–14. Adult zebrafish retinal neurons can successfully be cultured using our newly established protocol. Live cell images taken at DIV 1–14 in a Tg(Tru.gap43:GFP)^mil1(gap43) retinal cell culture demonstrate that GFP-expressing RGCs start to sprout neurites at DIV 1, and extensive networks are established from DIV 3 onwards. At DIV 5, clusters of retinal neurons start to form that increase in size during the following days in culture, as more and more neurons crowd together. As a result, cultures appear less evenly spread throughout the well. Time-lapse imaging discloses that these clusters are formed by active migration of retinal neurons (Supplementary Movie 2). Although neurons survive up to DIV 14, almost all neurons have grouped together at this point, and GFP expression is strongly decreased in RGC neurites. Immunostainings for the neurite marker Acetylated tubulin (magenta labeling, bottom panel) reveal healthy neurons with extensive neurites at DIV 14, indicating that the observed decrease in GFP labeling is due to the downregulation of the gap43 promoter, rather than dying of the cells. Scale bars: 100 μm. DIV, days in vitro; ONC, optic nerve crush; RGC, retinal ganglion cell.