Schematic representation of the experimental setup. (A) Timeline of the performed procedures. (B) Two days before culturing (DIV-2), fish are subjected to ONC to prime RGCs and improve their outgrowth in culture. In addition, MFDs are assembled by adhering the PDMS top part on a cleaned (acid treated) glass cover slip using plasma treatment. Next, MFDs are coated overnight with consecutively PLL (DIV-2) and Laminin (DIV-1). For both coatings, a volumetric gradient is used to ensure coating of the grooves. Additionally, a Laminin concentration gradient (from the AC to the SDC) is used to promote axonal outgrowth toward the AC. The next morning, adult zebrafish retinas are isolated, followed by an enzymatic and mechanical dissociation to obtain a single cell suspension. To remove dead cells and debris, the suspension is centrifuged and resuspended in fresh fish medium, and retinal cells are seeded in the SDC of an open compartment MFD (SOC450). To stimulate outgrowth of RGC axons toward the AC, a volumetric gradient from the SDC to the AC is created and conditioned retinal cells are also seeded in the AC. Cells are cultured at 30°C and 5% CO₂ and monitored daily. At DIV 2, RGC axonal outgrowth can be examined. At DIV 3, a vacuum-assisted axotomy is performed, whereafter axonal regeneration can be evaluated. To visualize the regenerative process and characterize mitochondrial dynamics or dendritic remodeling, respectively, Tg(Tru.gap43:GFP)^mil (gap43), Tg(Tru.gap43:mitoEGFP-2A-TagRFP-CAAX)^ulb17 (gapmito) or a sparsely-labeled, mixed culture of wild type (WT) and (gap43) retinal neurons are seeded in the SDC. AC, axonal compartment; DIV, days in vitro; MFD, microfluidic device; ONC, optic nerve crush; PLL, Poly-L-lysine; RGC, retinal ganglion cell; SDC, somatodendritic compartment.

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.

Adult zebrafish retinal cell culture in a microfluidic setup at DIV 3. (A) Adult zebrafish retinal neurons cultured in SDN450 MFDs, display limited outgrowth and network formation. Live cell images taken at DIV 3 demonstrate that Tg(Tru.gap43:GFP)^mil1 (gap43) retinal neurons, loaded in the wells and channel, do not attach properly into the channel, which causes a flow of neurons from the channel toward the wells after seeding (DIV 0) or addition of medium (DIV 0–3). As a result of this low density in the SDC channel, neurons display strongly reduced neurite outgrowth and network formation compared to neurons growing in the SDC wells, and only a few neurites grow into the microgrooves at DIV 3. (B) In contrast, at DIV 3, gap43 retinal neurons seeded in the SDC of an open compartment SOC450 MFD form an extensive neuronal network throughout the entire SDC, including the areas bordering the microgrooves. Consequently, spontaneous RGC outgrowth into the microgrooves is achieved in this setup, with numerous axons growing far into the AC at DIV 3. Scale bars: 500 μm (overview pictures), 100 μm (detailed pictures). AC, axonal compartment; CNB, complete neurobasal medium; DIV, days in vitro; MFD, microfluidic device; RGC, retinal ganglion cell; SDC, somatodendritic compartment.

Outgrowth of adult zebrafish RGC axons at DIV 1–3 in a microfluidic setup. Adult zebrafish retinal neurons can successfully be cultured in an open (SOC450) MFD. Confocal live cell images indicate that adult Tg(Tru.gap43:GFP)^mil1(gap43) zebrafish RGCs show early outgrowth and network formation from DIV 1 onwards, and that RGC axons are growing into the microgrooves and toward the AC at DIV 2. By DIV 3, numerous axons have reached to AC or are actively growing into the AC. Scale bars: 100 μm. AC, axonal compartment; CNB, complete neurobasal medium; DIV, days in vitro; MFD, microfluidic device; RGC, retinal ganglion cell; SDC, somatodendritic compartment.

Axotomy and regeneration of adult zebrafish RGCs at DIV 3–4. Representative confocal live cell microscopy images taken before, upon and after injury in the same section of the AC, disclose that adult zebrafish RGCs regenerate spontaneously upon in vitro axotomy in SOC450 MFDs. (A) At DIV3, when numerous adult Tg(Tru.gap43:GFP)^mil1 (gap43) zebrafish RGC axons are growing in the AC (left panels), an axotomy is performed using dual aspiration of all medium in the AC (DIV 3, 0 hpi, middle panels). One day later, at DIV 4 (24 hpi), numerous regenerating axons can be observed in the AC (regenerating axons, green arrowheads in right panels). However, some axons do not regenerate and stay stationary after axotomy (static axons, red arrowheads) or even degenerate upon injury (degenerating axons, blue arrowheads). Lastly, some axons, that did not reach the AC at the time of axotomy, further extend and form newly outgrowing axons that reach the AC in the hours following axotomy (0 hpi-24 hpi) (newly outgrowing axons, yellow arrowheads). These different states of axons can be distinguished using overview pictures at DIV 3 and DIV 4 in combination with time-lapse live imaging (Supplementary Movies 1, 5). (B) Representative live cell images taken at different time points after axotomy in the same section of the AC of a gap43 retinal culture after axotomy at DIV 3 reveal that regeneration is a dynamic process, and different axons extend at different moments and with different growth rates. Some pioneering axons start to regenerate immediately upon injury (0–1 hpi, *), while others only emerge after a few hours in the AC (1–3 hpi, ^×). Of note, occasionally axons make a U-turn and grow back into the microgrooves, rather than regrowing into the AC (purple arrowhead). Scale bar: 100 μm. AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.

Axotomy and regeneration of repeatedly injured adult zebrafish RGCs at DIV 3–5. Adult zebrafish RGC axons that have successfully sprouted after a first in vitro axotomy, regenerate again when a second in vitro axotomy is executed. Representative confocal live cell microscopy images taken before, upon and after injury in the same section of the AC of cultured Tg(Tru.gap43:GFP)^mil1(gap43) retinal neurons in an open compartment (SOC450) MFD disclose that 24 h after both the first (DIV 4, 24 hpi, 3th panels) and the second axotomy (DIV 5, 24 hpi2, 5th panels), regenerating (green arrowheads) as well as newly outgrowing (yellow arrowheads), static (red arrowheads) and degenerating axons (blue arrowheads) can be observed in the AC. These results indicate that repeated injuries (in vivo ONC, isolation, in vitro axotomy) do not negatively impact the spontaneous regenerative potential of cultured zebrafish RGCs. Of note, the high number of outgrowing and regenerating axons at DIV 5 complicates the correct identification of individual axons in the AC. Therefore, no further analyses should be performed on these cultures. Scale bar: 100 μm. AC, axonal compartment; DIV, days in vitro; hpi, hours post injury; MFD, microfluidic device; RGC, retinal ganglion cell.

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.

Characterization of changes in dendrite-like neurites during adult zebrafish RGC axonal regeneration. Time-lapse live imaging in sparesely labeled, mixed adult zebrafish retinal cultures seeded in a SOC450 MFD enables characterization of dendritic remodeling during axonal regeneration of adult zebrafish RGCs. Representative confocal still overview pictures of isolated gap43 RGCs (top panels) and detailed images of the dendritic area (bottom panels, with colored arrowheads indicating the same dendrite-like neurites over time and * indicating the axotomized axon) in a mixed Tg(Tru.gap43:GFP)^mil1/wild type (gap43/WT) retinal cell culture illustrate both axonal regeneration and temporary dendritic remodeling (in length and relative position) after axotomy at DIV 3 (0–3 hpi). These first evaluations indicate the potential of this sparsely-labeled setup to visualize and characterize dendritic changes during axonal regeneration of adult zebrafish RGCs. Scale bars: 100 μm (overview pictures); 50 μm (magnified inserts). AC, axonal compartment; DIV, days in vitro; MFD, microfluidic device; RGC, retinal ganglion cell; SDC, somatodendrtic compartment.

Visualization of mitochondria in adult zebrafish RGCs in microfluidic cultures. The recently developed Tg(Tru.gap43:mitoEGFP-2A-tagRFP-CAAX)^ulb17 (gapmito) reporter line enables to study mitochondrial motility in outgrowing and regenerating RGCs. (A) Confocal live cell images of gapmito retinal neurons at DIV 3 in an open compartment (SOC450) MFD indicate that outgrowing RGCs express RFP in membranes (middle panels) and EGFP in mitochondria in axons, dendrites and somata (bottom panels). (B) Detailed live cell images (left panels), as well as pictures of end-point DAPI-stained cultures of isolated neurons (right panels) in the SDC illustrate that mitochondria in the different neuronal compartments can be visualized with subcellular resolution. (C) Likewise in the microgrooves and AC, individual mitochondria can be identified in outgrowing gapmito RGC axons at DIV 3. These results demonstrate how the usefulness of this novel reporter line to characterize mitochondrial dynamics during axonal outgrowth or regeneration. Scale bars: 100 μm (A,C), 50 μm (B). AC, axonal compartment; DIV, days in vitro; MFD, microfluidic device; RGC, retinal ganglion cell; SDC, somatodendrtic compartment.

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.