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Axon growth enables the rapid wiring of the central nervous system. Understanding this process is a prerequisite to retriggering it under pathological conditions, such as a spinal cord injury, to elicit axon regeneration. The last decades saw progress in understanding the mechanisms underlying axon growth. Most of these studies employed cultured neurons grown on flat surfaces. Only recently studies on axon growth were performed in 3D. In these studies, physiological environments exposed more complex and dynamic aspects of axon development. Here, we describe current views on axon growth and highlight gaps in our knowledge. We discuss how axons interact with the extracellular matrix during development and the role of the growth cone and its cytoskeleton within. Finally, we propose that the time is ripe to study axon growth in a more physiological setting. This will help us uncover the physiologically relevant mechanisms underlying axon growth, and how they can be reactivated to induce axon regeneration., Competing Interests: Conflict of interest H. Witte, A. Ertürk, F. Hellal and F. Bradke filed a patent on the use of microtubule‐stabilising compounds for the treatment of lesion of CNS axons (European Patent no. 1858498; European patent application EP 11 00 9155.0; U.S. patent application 11/908,118). The authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.