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Tim McGinn, Dylan Leoni, Natalie Partington, Deborah L Stenkamp; Restoration of the dendritic fields and cone synapses of bipolar cells during retinal regeneration in the zebrafish. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1781. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Adult zebrafish have the capability to regenerate retinal neurons that have been lost due to damage. However, it is not known whether regenerated retinal neurons accurately “re-wire” themselves in the disrupted environment of a damaged retina, and this information is important for achieving the NEI’s Audacious Goal to regenerate neurons and neural connections in the eye. In this study we visualized regenerated retinal bipolar cells in zebrafish expressing YFP driven by the nyctalopin (nyx) promoter. We hypothesized that these bipolar cells would restore the sizes of their dendritic fields and restore connections with SWS2 (blue), RH2 (green), and LWS (red) cones over a 60 day time-course (when visually-mediated reflexes are regained).
Inner retinal neurons were destroyed by intraocular injection of ouabain. At 13, 17, 21, and 60 days post-injection (dpi), we imaged whole-mounted retinas containing nyx+ bipolar cells, SWS2, RH2, and LWS cones labeled with fluorescent reporter or antibody. We measured dendritic spread of nyx+ cells using the convex polygon method, and quantified their dendritic tips and patterns of cone connectivity.
Dendritic spread averaged 336µm2±239 (s.d.) in control retinas, 208µm2±142 (13 dpi), 115µm2±31 (17 dpi), 163µm2±80 (21 dpi), and 262µm2±178 (60 dpi) in lesioned retinas. Dendritic spread at 17 dpi was significantly reduced compared to controls (p<0.05, 1-way ANOVA, Bonferroni post-hoc). The average number of dendritic tips per nyx+ cell were 16.66±1.9 (s.d.) (controls), 13.29±8.3 (13 dpi), 15.33±11.0 (21 dpi), and 15.88± 4.9 (60 dpi) in lesioned retinas. At 17 dpi, many nyx+ cells lacked neurites with dendritic tips that could be quantified. The majority of analyzed nyx+ neurons made connections to SWS2 cones, and to RH2 and/or LWS cones in control, and 21 and 60 dpi retinas. At 13 and 17 dpi the majority of analyzed nyx+ neurons did not make connections to both SWS2 and to RH2 and/or LWS cones.
Our hypothesis was supported by the observed restoration of average dendritic spread of nyx+ bipolar cells, and of apparently normal cone connectivity patterns in regenerated retinas. The process of bipolar cell dendrite patterning appears dynamic during retinal regeneration at 13 and 17 dpi. Additional morphological and connectivity parameters are under analysis to characterize regenerating and regenerated nyx+ bipolar cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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