@article {69998, title = {Single-Particle Tracking of AMPA Receptor-Containing Vesicles}, journal = {Bio-protocol}, volume = {15}, year = {2025}, month = {2025 Jun 05}, abstract = {

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

{\textbullet} Track and characterize the motion of AMPAR GluA1+\ vesicles in cultured rat hippocampal neurons.

{\textbullet} GluA1 tagged with HaloTag (GluA1-HT) is expressed from native\ Gria1\ loci to avoid overexpression.

{\textbullet} 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).

{\textbullet} 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).

}, doi = {10.21769/BioProtoc.5325}, url = {https://doi.org/10.21769/BioProtoc.5325}, author = {Wong, Victor C. and Walpita, Deepika and Liu, Zhe and O{\textquoteright}Shea, Erin K} }