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 visu...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Bio-protocol LLC
2025-06-01
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| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5325&type=0 |
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| Summary: | 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. |
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| ISSN: | 2331-8325 |