Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse-Chase Labeling in Cultured Hippocampal Neurons.

The entanglement of long axons found in cultured dissociated hippocampal neurons restricts the analysis of the machinery underlying directed axonal trafficking. Further, hippocampal neurons exhibit "en passant" presynapses that may confound the analysis of long-range retrograde axonal transport. To solve these issues, we and others have developed microfluid-based methods to specifically follow the fates of the retrograde axonal cargoes following pulse-chase labeling by super-resolution live-cell imaging, and automatically tracking their directed transport and analyzing their kinetical properties. These methods have allowed us to visualize the trafficking of fluorescently tagged signaling endosomes and autophagosomes derived from axonal terminals and resolve their localizations and movements with high spatial and temporal accuracy. In this chapter, we describe how to use a commercially available microfluidic device to enable the labeling and tracking of retrograde axonal carriers, including (1) how to culture and transfect rat hippocampal neurons in the microfluidic device; (2) how to perform pulse-chase to label specific populations of retrograde axonal carriers; and (3) how to conduct the automatic tracking and data analysis using open-source software.