In Vivo Cytoskeletal <scp>AMPA</scp> Receptor Transport Imaging in <i>C. elegans</i>

Long-distance intracellular transport of ionotropic glutamate receptors (iGluRs) is essential for proper excitatory synaptic function underlying learning and memory. Many neuropsychiatric and neurodegenerative conditions have abnormal iGluR transport and trafficking, leading to an intense interest in the mechanisms and factors regulating these processes. Although iGluRs and synaptic protein transport have been studied in cultured neurons, in vitro systems lack the specific connectivity of native circuits essential for the organization and regulation of compartmentalized synaptic signaling. Here, we describe an in vivo imaging approach that leverages the optical transparency of C. elegans to measure the transport of glutamate receptors in a fully intact neural system. Our workflow includes a standardized protocol for worm mounting, high-resolution imaging, and quantification of motor-driven iGluR transport in C. elegans. We discuss critical parameters for optimal signal-to-noise ratio, analysis, and reproducibility. Through years of optimization, we have established which fluorophores and genetic tools are the most effective and reproducible for in vivo transport imaging. These results provide a refined and reproducible framework for studying motor-driven iGluR transport in an intact nervous system and highlight important technical variables that can affect in vivo transport imaging.