Background

The peripheral nervous system communicates sensory cues to the brain. It therefore mediates organismal responses to changes in the body’s internal and external environments (Handler and Ginty, 2021; Elias and Abdus-Saboor, 2022; Prescott and Liberles, 2022). Sensory neurons must maintain functionality throughout the life of the organism despite stress and trauma. Although acute loss of integrity of these neurons occurs persistently, animals normally overcome chronic neurological dysfunctions via effective repair (Johnson et al., 2005). By contrast, repair is normally extremely limited in the central nervous system (Varadarajan et al., 2022). Peripheral nerve wounding triggers dynamic changes in the behavior of Schwann cells as well as the recruitment of other cells including macrophages, which together resolve injury, initiate repair, and enhance regeneration (Abdo et al., 2019). In recent studies, we have combined genetic and chemical perturbations, transgenic markers, and high-resolution live imaging to characterize nerve injury and regeneration in larval zebrafish (Lozano-Ortega et al., 2018; Tian and López-Schier, 2020; Xiao et al., 2015a). Furthermore, we discovered that Schwann cells are important but not essential for sensory nerve regeneration (Tian et al., 2020b; Xiao et al., 2015b). Moreover, we revealed fast recruitment of macrophages to the wound (Tian et al., 2020). We have also discovered that blocking the degradation of axons severed from the neuronal cell body has no significant impact on nerve repair (Tian et al., 2020). Our finding that loss of the pro-degenerative protein Sarm1 accelerates axonal regeneration may have important implications for the development of therapies aimed at improving neural repair in humans.

The zebrafish larva is a powerful vertebrate experimental system for studies of neuronal wounding and regeneration (Rieger and Sagasti, 2011; Cardozo et al., 2017; Cigliola et al., 2020; González and Allende, 2021). Spatial and temporal control of injury can be done by laser-mediated axon transection or neuronal ablation. Axonal injury triggers fast degeneration of the part of the axon detached from the cell body, followed by regenerative growth of the proximal stump. In several previous studies, we used the mechanosensory lateral line in larval zebrafish to perform nerve wounding using an ultraviolet laser, and live microscopy in transgenic animals expressing fluorescent markers to track relevant cellular populations during injury resolution and repair (Figures 1 and 2). Of note, laser-mediated axon severing is a powerful technique but it suffers from the fact that both proximal and distal axon fragments are immediately cauterized. This is unlikely to occur during physical injury, when axoplasmic spillage may trigger different responses from the Schwann cells situated in proximity to the damaged nerve. Nevertheless, the precise spatiotemporal control of injury and simultaneous live imaging that this protocol allows vastly overcome this potential shortcoming. Therefore, we present a detailed protocol for facile implementation of laser-mediated microsurgery of sensory axons and in toto imaging of subsequent responses by axons, glia, and macrophages (Figure 3 and 4, and Videos 1 and 2). This protocol uses 5-day-post-fertilization (5 dpf) zebrafish Danio, but it can be equally used in older animals and other specimens of similar characteristics, including Oryzias, Astyanax, or Danionella.

An ultraviolet laser is directed to the target through a high numerical aperture objective lens mounted on a spinning-disk confocal microscope. However, a point-scanning single- or two-photon confocal microscope will be useful. Targeting is guided by the fluorescence from stable expression of EGFP or RFP using a lateralis afferent neuron-specific enhancer. However, mosaic expression of a fluorescent protein using pan-neuronal genetic control elements, for instance the widely employed NeuroD, Ngn1, or HuC, is also suitable.