Waller Thomas J, Smithson Laura J, Collins Catherine A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA
Cold Spring Harb Protoc. 2025 Jun 2;2025(6):pdb.prot108167. doi: 10.1101/pdb.prot108167.
Neurons extend their axons and dendrites over long distances and rely on evolutionarily conserved mechanisms to maintain the cellular structure and function of neurites at a distance from their cell body. Neurites that lose connection with their cell body following damage or stressors to their cytoskeleton undergo a programmed self-destruction process akin to apoptosis but using different cellular machinery, termed Wallerian degeneration. While first described for vertebrate axons by Augustus Waller in 1850, key discoveries of the enzymes that regulate Wallerian degeneration were made through forward genetic screens in Powerful techniques for genetic manipulation and visualization of single neurons combined with simple methods for introducing axotomy (neuron severing) to certain neuron types in have enabled the discovery and study of the cellular machinery responsible for Wallerian degeneration, in addition to mechanisms that enable clearance of the resulting debris. This protocol describes how to study the degeneration and clearance of axons from olfactory receptor neurons (ORNs). These peripheral neurons reside in the antennae and project axons to olfactory glomeruli of the anterior brain. Simple and nonlethal removal of antennae from adult flies causes axotomy of ORNs, and the fate of the injured axons can be readily visualized in a whole-mount dissected brain. This assay takes advantage of well-characterized genetic methods to robustly and specifically label subsets of ORNs. This method of neurite labeling and axotomy was the first axon injury paradigm to be developed in flies and is still regularly used due to its simplicity to perform, dissect, image, and analyze.
神经元将其轴突和树突延伸至很长的距离，并依靠进化上保守的机制来维持神经突在远离其细胞体处的细胞结构和功能。在其细胞骨架受到损伤或应激源作用后与细胞体失去连接的神经突会经历一种类似于细胞凋亡但使用不同细胞机制的程序性自我破坏过程，称为华勒氏变性。虽然1850年奥古斯塔斯·沃勒首次对脊椎动物轴突的华勒氏变性进行了描述，但调控华勒氏变性的酶的关键发现是通过正向遗传学筛选做出的。20世纪90年代，强大的单神经元遗传操作和可视化技术与向某些神经元类型引入轴突切断术（神经元切断）的简单方法相结合，除了发现了负责清除由此产生的碎片的机制外，还使得发现和研究负责华勒氏变性的细胞机制成为可能。本方案描述了如何研究嗅觉受体神经元（ORN）轴突的变性和清除。这些外周神经元位于触角中，并将轴突投射到前脑的嗅觉小球。从成年果蝇中简单且无致死性地去除触角会导致ORN轴突切断，并且在整个解剖的大脑中可以很容易地观察到受损轴突的命运。该实验利用了特征明确的遗传方法来有力且特异性地标记ORN的亚群。这种神经突标记和轴突切断的方法是在果蝇中开发的首个轴突损伤模型，并且由于其操作、解剖、成像和分析都很简单，至今仍经常被使用。
