Figure 7

Creation of a sparsely labeled mixed culture to visualize individual adult zebrafish RGCs. To enable characterization of dendritic remodeling during axonal outgrowth, a sparsely labeled, mixed zebrafish retinal cell culture is created. (A) Comparison of control Tg(Tru.gap43:GFP)^mil (gap43) and mixed gap43/wild type (gap43/WT) cultures at DIV 1 illustrate that seeding a mixture of cells at a ratio of 1:25 (gap43/WT) in the SDC of a SOC450 MFD results in a more sparsely labeled culture, without compromising cell density, which is essential to sustain outgrowth and network formation. Although most certainly a similar number of axons grow out in both conditions, in the mixed retinal cultures only a few labeled adult zebrafish RGCs become apparent at DIV 1 and can be seen growing into the microgrooves at DIV 2–3. (B) Confocal live imaging at DIV 3 demonstrates that in the sparsely labeled mixed cultures, individual RGCs, with axons growing into the microgrooves and AC, can be distinguished in the SDC. Scale bars: 100 μm. AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.