Optimized in vivo two-photon imaging reveals the essential role of the contralateral eye in functional optic nerve regeneration in zebrafish larvae.

Background: The visual pathway, consisting of the eye, optic nerve, and brain, serves as a valuable model for studying neural regeneration. The exceptional regenerative capacity of the zebrafish visual system enables detailed investigation of neural repair mechanisms in vivo. Although the transparency of zebrafish larvae permits real-time imaging of axonal regeneration following transection, previous methodological limitations such as pigment interference and suboptimal imaging protocols have hindered high-resolution analyses of structural recovery and cellular interaction throughout the entire visual pathway after optic nerve injury. This study aimed to overcome these barriers and enable comprehensive assessment of visual pathway regeneration.

Methods: In this study, we dissect the regenerative processes underlying structural recovery and cellular interplay across the entire visual pathway in larval zebrafish with an optic nerve transection model, using two-photon imaging and optokinetic response assays. Data were analyzed via multi-factorial ANOVA, unpaired t-tests, or Welch's t-test.

Results: We developed a longitudinal imaging platform by integrating two-photon microscopy (930 nm excitation), pigment suppression with phenylthiourea (PTU), and multi-axis positioning to observe visual pathway regeneration in vivo in zebrafish larvae at cellular resolution. This system enabled high-resolution imaging of the entire visual pathway, capturing the dynamics of green fluorescent protein (GFP)-labeled retinal ganglion cell (RGC) axons, optic nerve projections, and tectal reinnervation following optic nerve transection. Notably, enucleation of the contralateral eye resulted in aberrant optic nerve regrowth and impaired visual recovery after transection, indicating that guidance cues from the contralateral eye were essential for successful functional optic nerve regeneration. Additionally, the optimized two-photon imaging protocol allowed direct in vivo visualization of cellular interactions, revealing the rapid recruitment of DsRed-labeled neutrophils to the injured retina, optic nerve, and tectum during the repair process in double-transgenic Tg(lyz:DsRed); Tg(isl2b.2:Gal4-VP16; myl7:EGFP); Tg(4XnrUAS:GFP) larvae.

Conclusions: Our optimized imaging platform visualizes the entire visual pathway and cell interactions during regeneration, revealing contralateral eye is essential for functional recovery following optic nerve transection. Combined with multi-omics and calcium imaging, this approach potentially provides a powerful platform to decipher the cellular and molecular mechanisms of zebrafish eye-brain pathway reconstruction and offers insights into therapeutic targets for human optic neuropathies.