Single-Particle Tracking of AMPA Receptor-Containing Vesicles.

AMPA-type receptors are transported large distances to support synaptic plasticity at distal dendritic locations. Studying the motion of AMPA receptor vesicles can improve our understanding of the mechanisms that underlie learning and memory. Nevertheless, technical challenges that prevent the visualization of AMPA receptor vesicles limit our ability to study how these vesicles are trafficked. Existing methods rely on the overexpression of fluorescent protein-tagged AMPA receptors from plasmids, resulting in a saturated signal that obscures vesicles. Photobleaching must be applied to detect individual AMPA receptor vesicles, which may eliminate important vesicle populations from analysis. Here, we present a protocol to study AMPA receptor vesicles that addresses these challenges by 1) tagging AMPA receptors expressed from native loci with HaloTag and 2) employing a block-and-chase strategy with Janelia Fluor-conjugated HaloTag ligand to achieve sparse AMPA receptor labeling that obviates the need for photobleaching. After timelapse imaging is performed, AMPA receptor vesicles can be identified during image analysis, and their motion can be characterized using a single-particle tracking pipeline. Key features • Track and characterize the motion of AMPAR GluA1 vesicles in cultured rat hippocampal neurons. • GluA1 tagged with HaloTag (GluA1-HT) is expressed from native loci to avoid overexpression. • Sparse GluA1-HT labeling densities can be achieved without photobleaching via a block-and-chase strategy that utilizes Janelia Fluor (JF) dyes conjugated to HaloTag ligand (HTL). • GluA1-HT vesicles are identified during image analysis, and their motion is characterized using single-particle tracking (SPT) and hidden Markov modeling with Bayesian model selection (HMM-Bayes).