Optic nerve regeneration in larval zebrafish exhibits spontaneous capacity for retinotopic but not tectum specific axon targeting.

In contrast to mammals, retinal ganglion cells (RGC) axons of the optic nerve even in mature zebrafish exhibit a remarkable capacity for spontaneous regeneration. One constraint of using adult zebrafish is the limited ability to visualize the regeneration process in live animals. To dynamically visualize and trace the degree of target specific optic nerve regeneration, we took advantage of the optical transparency still preserved in post developmental larval zebrafish. We developed a rapid and robust assay to physically transect the larval optic nerve and find that by 96 hours post injury RGC axons have robustly regrown onto the optic tectum. We observe functional regeneration by 8 days post injury, and demonstrate that similar to adult zebrafish, optic nerve transection in larval zebrafish does not prominently induce cell death or proliferation of RGC neurons. Furthermore, we find that partial optic nerve transection results in axonal growth predominantly to the original, contralateral tectum, while complete transection results in innervation of both the correct contralateral and 'incorrect' ipsilateral tectum. Axonal tracing reveals that although regenerating axons innervate the 'incorrect' ipsilateral tectum, they successfully target their topographically appropriate synaptic areas. Combined, our results validate post developmental larval zebrafish as a powerful model for optic nerve regeneration, and reveal intricate mechanistic differences between axonal growth, midline guidance and synaptic targeting during zebrafish optic nerve regeneration.

Upregulation of leukemia inhibitory factor (LIF) during the early stage of optic nerve regeneration in zebrafish.

Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3-5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

Attempts to restore visual function after optic nerve damage in adult mammals.

Retinal ganglion cells (RGCs) and their axons, i.e., optic nerve (ON) fibers, provide a good experimental model for research on damaged CNS neurons and their functional ecovery. After the ON transection most RGCs undergo retrograde and anterograde degeneration but they can be rescued and regenerated by transplantation of a piece of peripheral nerve (PN). When the nerve graft was bridged to the visual center, regenerating RGC axons can restore the central visual projection. Behavioral recovery of relatively simple visual function has been proved in such PN-grafted rodents. Intravitreal injections of various neurotrophic factors and cytokines to activate intracellular signaling mechanism of RGCs and electrical stimulation to the cut end of ON have promoting effects on their survival and axonal regeneration. Axotomized RGCs in adult cats are also shown to survive and regenerate their axons through the PN graft. Among the cat RGC types, Y cells, which function as visual motion detector, tend to survive and regenerate axons better than others. X cells, which are essential for acute vision, suffer from rapid death after ON transection but they can be rescued by intravitreal application of neurotrophins accompanied with elevation of cAMP. To restore visual function in adult mammals with damaged optic pathway, the comprehensive and integrative strategies of multiple approaches will be needed, taking care of functional diversity of RGC types.