Dahlmanns M, Dahlmanns J (2022)
Publication Type: Book chapter / Article in edited volumes
Publication year: 2022
Publisher: Humana Press Inc.
Edited Volumes: Synaptic Vesicles
Series: Methods in Molecular Biology
City/Town: New York
Book Volume: 2417
Pages Range: 181-192
ISBN: 978-1-0716-1916-2
DOI: 10.1007/978-1-0716-1916-2_14
Synaptic vesicle exocytosis can be monitored with genetically encoded pH sensors in an in vitro fluorescence microscopy setup. Here, we describe a workflow starting with preparation of a primary cell culture to eventually estimate synaptic vesicle pool sizes based on electrical current-evoked vesicle release, which is reported by the synaptobrevin 2-EGFP fusion protein synapto-pHluorin (spH) that is expressed inside the synaptic vesicle membrane. The readily releasable pool and the recycling pool of synaptic vesicles are released separately in response to electrical stimulation. As vesicle reacidification is blocked in this experimental design, every released vesicle is counted only once. This spH-based approach offers different information than styryl-dye (FM dyes)-based approaches because the total synaptic pool size is measured by an alkalinization step. This provides a normalization constant for quantifying and comparing the synaptic vesicle pool sizes. In addition to investigation of basic research questions, spH-reported vesicle release is valuable to determine presynaptic effects of, e.g., pharmacological drug treatments.
APA:
Dahlmanns, M., & Dahlmanns, J. (2022). Synaptic Vesicle Pool Monitoring with Synapto-pHluorin. In Jana Dahlmanns, Marc Dahlmanns (Eds.), Synaptic Vesicles. (pp. 181-192). New York: Humana Press Inc..
MLA:
Dahlmanns, Marc, and Jana Dahlmanns. "Synaptic Vesicle Pool Monitoring with Synapto-pHluorin." Synaptic Vesicles. Ed. Jana Dahlmanns, Marc Dahlmanns, New York: Humana Press Inc., 2022. 181-192.
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