Targeted sensors for glutamatergic neurotransmission
Abstract
Optical report of neurotransmitter release allows visualization of excitatory synaptic transmission. Sensitive genetically-encoded glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, were enriched at synapses, retained function and reported spontaneous glutamate release in rat hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic mouse neurons cultured on astrocytic micro islands, evoked neurotransmitter release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal neurotransmitter release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short-term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.
Data availability
Custom software is available at GitHub.com/agplested/saft
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (390688087)
- Christian Rosenmund
- Andrew J R Plested
European Research Council (647895)
- Andrew J R Plested
Deutsche Forschungsgemeinschaft (323514590)
- Andrew J R Plested
Deutsche Forschungsgemeinschaft (446182550)
- Andrew J R Plested
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 housing and use were in compliance with, and approved by, the Animal Welfare Committee of Charité Medical University and the Berlin State Government Agency for Health and Social Services (Licenses T0220/09 and FMP_T 03/20). Newborn C57BLJ6/N mice (P0-P2) and Rats (P1-P3) of both sexes were used for all the experiments.
Copyright
© 2023, Hao 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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