Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Nobutoshi Harata; Timothy A Ryan; Stephen J Smith; JoAnn Buchanan; Richard W Tsien

doi:10.1073/pnas.171442798

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Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

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Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Nobutoshi Harata, Timothy A Ryan, Stephen J Smith, JoAnn Buchanan and Richard W Tsien

Proceedings of the National Academy of Sciences - PNAS, Vol.98(22), pp.12748-12753

10/23/2001

DOI: 10.1073/pnas.171442798

PMCID: PMC60125

PMID: 11675506

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Abstract

Exo–endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3′-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC−). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.

Biological Sciences

Details

Title: Subtitle: Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion
Creators: Nobutoshi Harata - Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345; and
Timothy A Ryan - Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345; and
Stephen J Smith - Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345; and
JoAnn Buchanan - Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345; and
Richard W Tsien - Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345; and
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.98(22), pp.12748-12753
DOI: 10.1073/pnas.171442798
PMID: 11675506
PMCID: PMC60125
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: The National Academy of Sciences
Language: English
Date published: 10/23/2001
Academic Unit: Molecular Physiology and Biophysics
Record Identifier: 9984025407702771

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