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 Gria1 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).