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.

与哺乳动物不同，成熟斑马鱼的视神经视网膜神经节细胞（retinal ganglion cells, RGC）轴突具备极强的自发性再生能力。使用成年斑马鱼开展研究的一大局限，是难以在活体动物中实时可视化再生过程。为了动态可视化并追踪靶标特异性视神经再生的程度，我们利用了发育后期幼龄斑马鱼仍保留的光学透明特性。我们开发了一种快速且稳定的实验方法，可对幼龄斑马鱼的视神经进行物理截断，并发现损伤后96小时，RGC轴突已大量再生至视顶盖（optic tectum）。我们在损伤后8天观察到了功能性再生，并证实与成年斑马鱼的情况一致，幼龄斑马鱼的视神经截断不会显著诱导RGC神经元发生细胞死亡或增殖。此外，我们发现部分视神经截断会导致轴突主要生长至原始的对侧（contralateral）视顶盖，而完全视神经截断则会使轴突同时支配正确的对侧视顶盖与“错误的”同侧（ipsilateral）视顶盖。轴突追踪实验显示，尽管再生轴突会支配“错误的”同侧视顶盖，但它们仍能精准靶向其拓扑适配的突触区域。综上，我们的研究结果验证了发育后期幼龄斑马鱼可作为视神经再生研究的强大模型，并揭示了斑马鱼视神经再生过程中轴突生长、中线导向与突触靶向之间复杂的机制差异。