Abstract
We previously reported strikingly normal morphologies and functional connectivities of regenerated retinal bipolar neurons (BPs) in zebrafish retinas sampled 60 days after a ouabain-mediated lesion of inner retinal neurons (60 DPI) (McGinn et al., 2018). Here we report early steps in the birth of BPs and formation of their dendritic trees and axonal arbors during regeneration. Adult zebrafish were subjected to ouabain-mediated lesion that destroys inner retinal neurons but spares photoreceptors and Müller glia, and were sampled at 13, 17, and 21 DPI, a timeframe over which plexiform layers reemerge. We show that this timeframe corresponds to reemergence of two populations of BPs (PKCα+ and nyx::mYFP+). Sequential BrdU, EdU incorporation reveals that similar fractions of PKCα+ BPs and HuC/D+ amacrine/ganglion cells are regenerated concurrently, suggesting that the sequence of neuronal production during retinal regeneration does not strictly match that observed during embryonic development. Further, accumulation of regenerated BPs appears protracted, at least through 21 DPI. The existence of isolated, nyx::mYFP+ BPs allowed examination of cytological detail through confocal microscopy, image tracing, morphometric analyses, identification of cone synaptic contacts, and rendering/visualization. Apically-projecting neurites (=dendrites) of regenerated BPs sampled at 13, 17, and 21 DPI are either truncated, or display smaller dendritic trees when compared to controls. In cases where BP dendrites reach the outer plexiform layer (OPL), numbers of dendritic tips are similar to those of controls at all sampling times. Further, by 13–17 DPI, BPs with dendritic tips reaching the outer nuclear layer (ONL) show patterns of photoreceptor connections that are statistically indistinguishable from controls, while those sampled at 21 DPI slightly favor contacts with double cone synaptic terminals over those of blue-sensitive cones. These findings suggest that once regenerated BP dendrites reach the OPL, normal photoreceptor connectomes are established, albeit with some plasticity. Through 17 DPI, some basally-projecting neurites (=axons) of regenerated nyx::mYFP+ BPs traverse long distances, branch into inappropriate layers, or appear to abruptly terminate. These findings suggest that, after a tissue-disrupting lesion, regeneration of inner retinal neurons is a dynamic process that includes ongoing genesis of new neurons and changes in BP morphology.
Highlights
A major challenge in the treatment of human neurodegenerative diseases and trauma affecting the central nervous system is that mammals do not regenerate neurons lost to such conditions, but instead launch a gliotic response that results in tissue scarring (Pekny et al, 2014; Okada et al, 2018), and nervous tissue function is not restored
We previously documented strikingly normal morphologies of regenerated bipolar neurons (BPs) in retinas sampled at 60 days after a ouabainmediated lesion of inner retinal neurons (McGinn et al, 2018)
We reasoned that 13–17 days post-injury (DPI) may represent a period of BP reemergence as well as synaptogenesis and refinement of neuronal processes during retinal regeneration
Summary
A major challenge in the treatment of human neurodegenerative diseases and trauma affecting the central nervous system is that mammals do not regenerate neurons lost to such conditions, but instead launch a gliotic response that results in tissue scarring (Pekny et al, 2014; Okada et al, 2018), and nervous tissue function is not restored. Several regulatory factors have been identified that promote or are necessary for Müller glia to re-enter the cell cycle and generate neural progenitors (Fausett et al, 2008; Qin et al, 2011; Ramachandran et al, 2011a,b; Lenkowski et al, 2013; Nagashima et al, 2013; Nelson et al, 2013), which proliferate and produce the regenerated retinal neurons (Nagashima et al, 2013; Gorsuch and Hyde, 2014; Powell et al, 2016). Some of this knowledge has already been applied in strategies for coaxing the Müller glia of mouse retina to generate retinal neurons (Ahmad et al, 2011; Hyde and Reh, 2014; Jorstad et al, 2017; Yao et al, 2018)
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