Individual or grouped cells, spatially isolated, can undergo in-depth gene expression analysis using the effective LCM-seq technology. Within the retina's visual system, the retinal ganglion cell layer is the specific location of the retinal ganglion cells (RGCs), which serve as the eye-brain connection through the optic nerve. A uniquely advantageous location facilitates RNA retrieval via laser capture microdissection (LCM) from a substantially enriched cell population. The application of this method allows for the study of extensive modifications in gene expression within the transcriptome subsequent to injury to the optic nerve. Within the zebrafish model, this methodology reveals the molecular drivers of successful optic nerve regeneration, standing in stark contrast to the inability of mammalian central nervous systems to regenerate axons. This approach outlines how to find the least common multiple (LCM) within various zebrafish retinal layers, after optic nerve damage, and while the optic nerve is regenerating. RNA extracted using this protocol is adequate for RNA-Seq library preparation and subsequent analysis.

Recent improvements in technical methods have facilitated the separation and purification of mRNAs from diverse genetic cell types, allowing for a more encompassing view of gene expression related to gene regulatory networks. These instruments provide the capability to compare the genome of organisms undergoing a variety of developmental or diseased states and environmental or behavioral conditions. Using transgenic animals harboring a ribosomal affinity tag (ribotag), the TRAP method facilitates rapid isolation of distinct genetically labeled cell populations, which are targeted to ribosome-bound mRNAs. In this chapter, we furnish a progressively detailed methodology for implementing a revised TRAP protocol in Xenopus laevis, the South African clawed frog. Also included is an explanation of the experimental design, focusing on the necessary controls and their justifications, combined with a detailed breakdown of the bioinformatic procedures for analyzing the Xenopus laevis translatome using TRAP and RNA-Seq.

The recovery of function, within days after spinal injury, in larval zebrafish, is marked by axonal regrowth over a complex injury site. We outline a simple protocol for disrupting gene function in this model by using acute injections of highly active synthetic guide RNAs. This approach facilitates the rapid detection of loss-of-function phenotypes without resorting to breeding.

Axon damage brings about a complex array of outcomes, incorporating successful regeneration and the reinstatement of normal function, the failure of regeneration, or the demise of the neuron. Through experimental injury of an axon, the degenerative process of the detached distal segment from the cell body can be investigated, and the subsequent stages of regeneration can be documented. PGE2 in vivo Environmental damage around an axon is minimized by precise injury, thereby reducing the involvement of extrinsic factors like scarring or inflammation. This approach facilitates isolation of the regenerative role of intrinsic components. Different approaches have been taken to section axons, each with its own set of advantages and disadvantages inherent to the method. Employing a laser in a two-photon microscope, this chapter describes severing individual axons of touch-sensing neurons in zebrafish larvae, and live confocal imaging for monitoring the regeneration process, which provides exceptional resolution.

Following an injury, axolotls exhibit the capacity for functional spinal cord regeneration, recovering both motor and sensory function. In contrast to other responses, severe spinal cord injuries in humans are countered by the formation of a glial scar. This scar, while effective in preventing further damage, also hinders any regenerative processes, thereby leading to functional loss caudal to the injury. Axolotls are now widely used to dissect the cellular and molecular events that contribute to the remarkable capacity for successful central nervous system regeneration. Experimental axolotl injuries, such as tail amputation and transection, do not mirror the prevalent blunt force trauma suffered by humans. This research describes a more clinically relevant spinal cord injury model in the axolotl, using a weight-drop methodology. The drop height, weight, compression, and injury position are all precisely controllable parameters of this reproducible model, allowing for precise determination of the injury's severity.

Injury to zebrafish retinal neurons does not prevent functional regeneration. Lesions affecting specific neuronal cell populations, along with photic, chemical, mechanical, surgical, and cryogenic lesions, are followed by the regenerative process. Studies on regeneration using chemical retinal lesions are aided by the broad, expansive, and geographically widespread nature of the lesion. The loss of visual function is compounded by a regenerative response that engages nearly all stem cells, prominently Muller glia. These lesions are therefore instrumental in expanding our knowledge of the underlying processes and mechanisms involved in the re-creation of neuronal pathways, retinal functionality, and visually stimulated behaviours. Widespread chemical retinal lesions enable quantitative gene expression analysis, from initial damage to complete regeneration, allowing a study of regenerated retinal ganglion cell axons' growth and targeting. Ouabain, a neurotoxic Na+/K+ ATPase inhibitor, surpasses other chemical lesions in its inherent scalability. The extent of damage, whether it encompasses only inner retinal neurons or involves all retinal neurons, is readily adjustable through variations in the utilized intraocular ouabain concentration. We describe the method used to generate selective or extensive retinal lesions.

Human optic neuropathies are a source of debilitating conditions, leading to the loss of vision, either partially or completely. Within the intricate structure of the retina, retinal ganglion cells (RGCs) are the only cell type that provides the cellular link between the visual input of the eye and the brain. RGC axon damage within the optic nerve, while sparing the nerve's sheath, represents a model for both traumatic optical neuropathies and progressive conditions like glaucoma. This chapter explores two varying surgical methods for the creation of an optic nerve crush (ONC) in the post-metamorphic frog, Xenopus laevis. Why is the frog a valuable subject in the realm of biological modeling? The capacity for regenerating damaged central nervous system neurons, present in amphibians and fish, is absent in mammals, leaving them unable to regenerate retinal ganglion cell bodies and axons after injury. Two distinct surgical approaches to ONC injury are presented, followed by an assessment of their respective strengths and limitations. We also explore the unique features of Xenopus laevis as a model organism for examining CNS regeneration.

Regeneration of the zebrafish's central nervous system is a remarkable and spontaneous capacity. Due to their optical transparency, larval zebrafish are frequently utilized for observing cellular processes in live animals, like nerve regeneration. Previous research on the regeneration of RGC axons within the optic nerve has involved adult zebrafish. Unlike prior studies, this research will evaluate optic nerve regeneration in larval zebrafish. Taking advantage of the imaging resources available in larval zebrafish models, we recently developed an experimental approach to physically sever RGC axons and observe the regeneration of their optic nerves within these larval zebrafish. Our findings indicated that RGC axons regenerated to the optic tectum in a rapid and robust manner. Procedures for optic nerve transections and visualization of retinal ganglion cell regeneration in larval zebrafish are presented in this document.

Central nervous system (CNS) injuries, as well as neurodegenerative diseases, often exhibit axonal damage alongside dendritic pathology. Zebrafish, unlike mammals, display a robust regeneration capability within their central nervous system (CNS) after injury, making them an ideal model to further unravel the processes driving axonal and dendritic regrowth. We start by describing, in adult zebrafish, an optic nerve crush injury model, a paradigm which causes both the degeneration and regrowth of retinal ganglion cell axons (RGCs), but also initiates a patterned and scheduled breakdown and subsequent recovery of RGC dendrites. Next, we provide detailed protocols for measuring axonal regeneration and synaptic reinstatement in the brain, utilizing retro- and anterograde tracing experiments, complemented by immunofluorescent staining of presynaptic compartments. Finally, a detailed description of methods for the analysis of RGC dendrite retraction and subsequent regrowth within the retina is provided, incorporating morphological measurements and immunofluorescent staining for dendritic and synaptic markers.

Protein expression, regulated spatially and temporally, is essential for various cellular functions, particularly in highly polarized cells. While protein relocation from other cellular compartments can modify the subcellular proteome, transporting messenger RNA to specific subcellular locations allows for localized protein synthesis in response to various stimuli. Dendrite and axon elongation within neurons is intricately tied to the spatial specificity of protein synthesis, which occurs in regions distant from the neuronal cell body. PGE2 in vivo This presentation of developed methodologies for localized protein synthesis is anchored by the example of axonal protein synthesis. PGE2 in vivo Employing dual fluorescence recovery after photobleaching, we delineate protein synthesis sites in detail, using reporter cDNAs that encode two different subcellular location mRNAs paired with diffusion-limited fluorescent reporter proteins. We demonstrate the method's capacity to track, in real-time, alterations in the specificity of local mRNA translation prompted by extracellular stimuli and varying physiological states.