LCM-seq's potent capability in gene expression analysis extends to spatially separated groups or individual cells. 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 precisely delineated site presents a singular chance to collect RNA using laser capture microdissection (LCM) from a richly concentrated cellular population. This approach permits a comprehensive investigation of transcriptome-wide shifts in gene expression patterns in the wake of optic nerve injury. Utilizing the zebrafish model, this approach discerns molecular events responsible for successful optic nerve regeneration, unlike the mammalian central nervous system's inability to regenerate axons. The least common multiple (LCM) from various zebrafish retinal layers is determined using a method, after optic nerve damage and throughout optic nerve regeneration. For RNA-Seq or subsequent experimental analysis, the RNA purified using this method is satisfactory.
Innovative technical procedures now permit the isolation and purification of mRNAs from genetically distinct cell types, providing a more comprehensive overview of gene expression and its relationship to gene networks. These tools enable researchers to compare the genome profiles of organisms encountering diverse developmental, disease, environmental, and behavioral conditions. Transgenic animals expressing a ribosomal affinity tag (ribotag) are used in the TRAP (Translating Ribosome Affinity Purification) method to efficiently isolate genetically different cell populations, focusing on mRNAs associated with ribosomes. We present, in this chapter, an updated and stepwise procedure for performing the TRAP method on the South African clawed frog, Xenopus laevis. A comprehensive overview of the experimental plan, particularly the critical controls and their reasoning, and the detailed bioinformatic steps for analyzing the Xenopus laevis translatome using TRAP and RNA-Seq, is also presented.
Zebrafish larvae successfully regenerate axons across a complex spinal injury site, leading to the restoration of function in just a few days. A straightforward protocol for disrupting gene function in this model is detailed here, using swift injections of potent synthetic gRNAs to quickly ascertain loss-of-function phenotypes without the requirement for breeding.
Axon sectioning yields varied consequences, ranging from successful regeneration and the reinstatement of function to a failure in regeneration, or even neuronal cell death. An axon's experimental injury allows for the examination of the degenerative pathway in the distal segment, separated from the cell body, and the documentation of the regeneration sequence. learn more Precisely targeted injury to an axon minimizes damage to the surrounding environment, thereby limiting the influence of extrinsic processes such as scarring and inflammation. Consequently, researchers can better isolate the intrinsic regenerative factors at play. Diverse techniques for severing axons have been implemented, each with its own inherent advantages and disadvantages. Zebrafish larval touch-sensing neuron axons are precisely severed using a laser within a two-photon microscope, while live confocal imaging monitors their regeneration in real-time; this method provides a uniquely high resolution.
Following an injury, axolotls exhibit the capacity for functional spinal cord regeneration, recovering both motor and sensory function. Unlike other responses, severe spinal cord injury in humans triggers the formation of a glial scar. This scar, though protective against further damage, obstructs regenerative processes, resulting in functional impairment in the spinal cord regions below the injury. Successful central nervous system regeneration, in the axolotl, provides a valuable framework for understanding the interplay of cellular and molecular events. Although tail amputation and transection are used in axolotl experiments, they do not effectively simulate the blunt trauma common in human injuries. We present, in this report, a more clinically applicable model for spinal cord injuries in the axolotl, employing a weight-drop method. By precisely controlling the drop height, weight, compression, and impact position, this replicable model meticulously adjusts the severity of the incurred harm.
Following injury, zebrafish successfully regenerate functional retinal neurons. Photic, chemical, mechanical, surgical, cryogenic lesions, and those specifically impacting neuronal populations, are all conditions followed by regeneration. One significant advantage of chemically induced retinal lesions in regeneration studies is their broad and widespread topographical effect. Visual impairment is a direct outcome, accompanied by a regenerative response that mobilizes nearly all stem cells, particularly Muller glia. Subsequently, these lesions facilitate a greater comprehension of the procedures and mechanisms enabling the re-establishment of neural connections, retinal performance, and actions influenced by visual perception. Gene expression throughout the retina, during both the initial damage and regeneration periods, can be quantitatively assessed using widespread chemical lesions. This also allows for investigation into the growth and axonal targeting of regenerated retinal ganglion cells. The unique characteristic of ouabain, a neurotoxic Na+/K+ ATPase inhibitor, lies in its scalability, an advantage not shared by other chemical lesions. The selective damage to retinal neurons, encompassing either just the inner layers or all retinal neurons, depends entirely on the intraocular ouabain concentration. We describe the method used to generate selective or extensive retinal lesions.
Human optic neuropathies frequently trigger incapacitating conditions, leading to either partial or total vision impairment. Despite the retina's multifaceted cellular structure, retinal ganglion cells (RGCs) represent the only cellular pathway that transmits information from the eye to the brain. Progressive neuropathies, including glaucoma, and traumatic optical neuropathies share a common model: optic nerve crush injuries which cause damage to RGC axons but spare the nerve sheath. This chapter describes two unique surgical approaches for the creation of an optic nerve crush (ONC) in post-metamorphic Xenopus laevis frogs. What are the justifications for selecting the frog as an experimental model? The inability of mammals to regenerate damaged central nervous system neurons, including retinal ganglion cells and their axons, stands in stark contrast to the regenerative capacity of amphibians and fish. We not only present two contrasting surgical ONC injury techniques, but also analyze their strengths and weaknesses, and delve into the particular characteristics of Xenopus laevis as a biological model for studying central nervous system regeneration.
Zebrafish have an extraordinary capability for the spontaneous restoration of their central nervous system. The inherent optical transparency of zebrafish larvae makes them ideal for live-animal observation of cellular processes, such as nerve regeneration. In the past, adult zebrafish models have been employed to investigate the regeneration of RGC axons in the optic nerve. Prior studies have not explored optic nerve regeneration in larval zebrafish specimens; this study addresses this gap. Our recent development of an assay in the larval zebrafish model is designed to physically transect RGC axons and observe subsequent optic nerve regeneration, taking full advantage of the imaging capacities within these organisms. Our findings indicated that RGC axons regenerated to the optic tectum in a rapid and robust manner. This work describes the techniques for optic nerve transections in larval zebrafish, as well as methods for visualizing retinal ganglion cell regrowth.
Dendritic pathology, often concurrent with axonal damage, is a common feature of central nervous system (CNS) injuries and neurodegenerative diseases. Adult zebrafish, unlike mammals, exhibit a strong regeneration capability in their central nervous system (CNS) after injury, making them a valuable model organism for understanding the mechanisms driving axonal and dendritic regrowth following CNS damage. We first detail an optic nerve crush injury model in adult zebrafish, a procedure that causes de- and regeneration of retinal ganglion cell (RGC) axons, coupled with the precise and predictable disintegration, and subsequent restoration of RGC dendrites. We subsequently detail the methodologies for assessing axonal regrowth and synaptic re-establishment within the brain, employing retro- and anterograde tracing techniques and immunofluorescent staining procedures targeting presynaptic components. 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. Relocating proteins from different cellular domains can alter the subcellular proteome, whereas the transport of mRNAs to subcellular regions permits localized protein synthesis in response to changing circumstances. Neurons rely on localized protein synthesis—a crucial mechanism—to generate and extend dendrites and axons significantly from the parent cell body. learn more To investigate localized protein synthesis, this discussion utilizes axonal protein synthesis as a case study, exploring the developed methodologies. learn more To visualize protein synthesis sites, a meticulous dual fluorescence recovery after photobleaching technique was employed, which utilizes reporter cDNAs encoding two unique localizing mRNAs alongside diffusion-limited fluorescent reporter proteins. This method reveals how extracellular stimuli and different physiological states dynamically modify the specificity of local mRNA translation, tracked in real-time.