Probing vesicle dynamics in single hippocampal synapses.
We use fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study vesicle dynamics inside the synapses of cultured hippocampal neurons labeled with the fluorescent vesicle marker FM 1-43. These studies show that when the cell is electrically at rest, only a small population of vesicles is mobile, taking seconds to traverse the synapse. Applying the phosphatase inhibitor okadaic acid causes vesicles to diffuse freely, moving 30 times faster than vesicles in control synapses. These results suggest that vesicles move sluggishly due to binding to elements of the synaptic cytomatrix and that this binding is altered by phosphorylation. Motivated by these results, a model is constructed consisting of diffusing vesicles that bind reversibly to the cytomatrix. This stick-and-diffuse model accounts for the fluorescence correlation spectroscopy and fluorescence recovery after photobleaching data, and also predicts the well-known exponential refilling of the readily releasable pool. Our measurements suggest that the movement of vesicles to the active zone is the rate-limiting step in this process.
Shtrahman
Matthew
M
Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Yeung
Chuck
C
Nauen
David W
DW
Bi
Guo-qiang
GQ
Wu
Xiao-Lun
XL
D000818:Animals; D001703:Biophysics / Q000379:methods*; D002478:Cells, Cultured; D004058:Diffusion; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D006624:Hippocampus / Q000166:cytology* / Q000196:embryology / Q000473:pathology*; D008954:Models, Biological; D015233:Models, Statistical; D009474:Neurons / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013569:Synapses / Q000378:metabolism*; D013997:Time Factors
D004791:Enzyme Inhibitors; D019319:Okadaic Acid; D010744:Phosphoric Monoester Hydrolases