Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor
- Vanderbilt University, United States
Abstract
Synapses maintain both action potential-evoked and spontaneous neurotransmitter release, however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In non-neuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of non-photobleached probes with a time constant of 10 seconds. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files; Source Data files have been provided for all Figures and Figure Supplements. The custom Matlab script used to analyse the data is deposited in GitHub (https://github.com/camilleswang/iGluSnFR-Analysis) and is freely available.
Article and author information
Author details
Funding
National Institute of Mental Health (MH66198)
- Ege T Kavalali
National Institute of Mental Health (MH081060)
- Lisa M Monteggia
National Institute of Mental Health (MH070727)
- Lisa M Monteggia
National Institute of General Medical Sciences (GM007347)
- Camille S Wang
Brain and Behavior Research Foundation
- Natali L Chanaday
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal experimentation: Animal procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Vanderbilt University School of Medicine (Animal Protocol Number M1800103).
Copyright
© 2022, Wang et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor
eLife 11:e76008.
https://doi.org/10.7554/eLife.76008
