Figure 10

Characterization of mitochondrial motility during axonal outgrowth and regeneration of adult zebrafish RGCs. To characterize mitochondrial dynamics in outgrowing and regenerating RGCs, kymographs of time-lapse live movies, acquired in adult zebrafish retinal neurons cultured in an open compartment (SOC450) MFD, have been generated. Representative confocal still images from time-lapse live recordings in Tg(Tru.gap43:mitoEGFP-2A-tagRFP-CAAX)^ulb17 (gapmito) RGC axons and corresponding kymographs illustrate mitochondrial mobility during axonal outgrowth (DIV 2), and in regenerating, static (non-regenerating) and degenerating axons at different time points after axotomy (DIV 3 0–5 hpi). On each kymograph, stationary (vertical lines) as well as motile mitochondria (diagonal lines) can be observed, with mitochondria moving both in anterograde and retrograde directions. Moreover, in outgrowing and regenerating axons, an accumulation of mitochondria can be observed at the distal end of the axon, presumably in the growth cones (blue arrowheads). Of note, these kymographs can also give some first indications on the occurence of additional mitochondrial dynamics like fission (red arrowheads) or fusion (green arrowheads). Kymographs are generated using ImageJ for 20–30 min with an 5-s interval. Scale bars: 10 min (vertical) and 10 μm (horizontal). AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.