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The axonal microtubules (MTs) support long-distance transport of cargoes that are dispatched to distinct cellular subcompartments. Among them, mRNAs are directly transported in membraneless ribonucleoprotein (RNP) granules that, together with ribosomes, can also hitchhike on fast-moving membrane-bound organelles for accurate transport along MTs. These organelles serve as platforms for mRNA translation, thus generating axonal foci of newly synthesized proteins. Local translation along axons not only supports MT network integrity but also modulates the processivity and function of molecular motors to allow proper trafficking of cargoes along MTs. Thus, identifying the mechanisms that coordinate axonal transport with local protein synthesis will shed new light on the processes underlying axon development and maintenance, whose deregulation often contribute to neurological disorders.

Summary Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment. For complete details on the use and execution of this protocol, please refer to Even et al. (2019)., Graphical Abstract, Highlights • Descriptive method to electroporate DNA plasmids in the embryonic mouse cortex • Step-by-step procedure to generate and mount organotypic brain slices • Protocol to record and analyze axonal transport in callosal projection neurons • Guidelines for protocol troubleshooting and overview on its limitations, Axonal transport is a physiological process adopted by neurons to transport organelles, proteins, and other molecules along their axonal projections. Here, we describe a step-by-step protocol to record the dynamics of axonal transport along the projections of callosal neurons by combining the in utero electroporation technique with the preparation of postnatal organotypic cortical slices. This ex vivo protocol has been developed to investigate axonal transport in a physiological setting closely reproducing the in vivo environment.