Targeted sensors for glutamatergic neurotransmission
- Institute of Biology, Cellular Biophysics, Humboldt-Universität zu Berlin, Germany
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Germany
- Institute for Neurophysiology, Charité - Universitätsmedizin Berlin, Germany
- NeuroCure Cluster of Excellence, Germany
Figures
Figure 1 with 3 supplements
Patch-clamp fluorometry of SnFR-γ2 and SnFR-γ8 in HEK cells.
(A) Schematic view of SnFR-γ2 and SnFR-γ8 chimeras, comprised of the truncated extracellular part of iGluSnFR (orange) to signal glutamate binding (black star), NETO as transmembrane linker (green) …
Figure 1—figure supplement 1
Kinetic properties and selectivity of iGluSnFR variants in HEK cells.
(A) Patch-clamp fluorometry of iGluSnFR (orange traces) and the SnFR-Y230F variant (blue traces) in whole lifted cells revealed a stronger fluorescence response and faster off kinetics for Y230F …
Figure 1—figure supplement 2
Evoked currents and fluorescence signals of iGluSnFR, SnFR-Y230F variant, and synapto-pHluorin on autaptic neurons.
(A) Representative fluorescence micrograph (scale bar, 5 μm) for an autaptic neuron expressing iGluSnFR with a region of interest (ROI) marked. (B) Fluorescence time series from the ROI recorded …
Figure 1—figure supplement 3
Poor optical performance of truncated SnFR insertions to chimeric AMPA receptors.
(A) Engineering of GluA—iGluSnFR chimeras. Truncated iGluSnFR (GltI and cpGFP), flanked by short linker sequences was inserted at 383 and DAQ sites at the beginning of the linker between ATD and LBD …
Figure 2
Spontaneous responses of iGluSnFR, SnFR-γ2, and SnFR-γ8 expressed in cultured rat hippocampal neurons.
(A) Fluorescence micrograph of neurons expressing iGluSnFR with three typical regions of interest marked (ROIs, indicated as magenta circles) and compared with adjacent ROIs of the same size (shown …
Figure 3
Localisation of iGluSnFR variants at synapses.
(A) Representative images of hippocampal neurons infected with iGluSnFR, SnFR-γ2, and SnFR-γ8 and live immunostained with antibodies against GFP for surface labelling. After fixation, cells were …
Figure 4
SnFR-γ2 and SnFR-γ8 give a more spatially precise signal than iGluSnFR.
(A) Time-lapse with 40-Hz frame rate of representative spontaneous fluorescence responses for iGluSnFR and SnFR-γ2 (separate recordings from separate neurons). For SnFR-γ2, the signal coincides with …
Figure 5 with 2 supplements
Early infection with SnFR-γ2 or Stargazin disrupts glutamatergic currents in autaptic hippocampal neurons.
(A) Responses of autaptic neurons infected with SnFR-γ2 or iGluSnFR to a five pulse train at 2 Hz. (B) The maximum projections of the fluorescence Eilers and Boelens, 2005 from each cell in panel (A)…
Figure 5—figure supplement 1
Neither SnFR-γ2 nor SnFR-γ8 influences synapse density or size in hippocampal neuron cultures.
(A) Representative images of synapse density in control (WT), iGluSnFR, SnFR-γ2, and SnFR-γ8 expressed in bulk hippocampal neuron cultures infected at DIV 1–3 (middle panel) or DIV 6 (right panel). …
Figure 5—figure supplement 2
Electrophysiological parameters of autapses overexpressing Stargazin constructs.
Miniature excitatory postsynaptic current (EPSC) amplitude (A), mEPSC frequency (B), and paired-pulse ratio (P.P.R.) (C) for neurons expressing mScarlet-Stg (green) or mScarlet-NETO-Stg (magenta) …
Figure 6
Evoked currents and fluorescent responses of iGluSnFR and SnFR-γ2 from autaptic neurons.
(Α) A train of five action potentials (APs) evoked five glutamatergic currents (excitatory postsynaptic currents, EPSCs) from an autaptic neuron infected with iGluSnFR (infected after DIV 6) in 2 mM …
Figure 7 with 2 supplements
Targeted sensors give smaller and more reliable regions of interest (ROIs).
(A) ROI size analysis for evoked fluorescent responses of SnFR, SnFR-γ2, and SnFR-γ8 οn autaptic neurons. ROIs (orange outlines) automatically selected by a custom-written Fiji script (Source code 1)…
Figure 7—figure supplement 1
Illustrative analysis of rundown of the fluorescence signal.
(A) The smoothed amplitudes of 50 peaks extracted from movies of two neurons, one expressing iGluSnFR and one expressing SnFR-γ2. Forty-five regions of interest (ROIs) were extracted for SnFR and 22 …
Figure 7—figure supplement 2
Comparing rundown between electrophysiological responses and optical report.
(A) Stable baseline fluorescence intensities (in arbitrary units, AU) for individual regions of interest (ROIs) in a single-cell expressing iGluSnFR (left) and one expressing SnFR-γ2 (right). …
Figure 8
Correlations and variance of fluorescence responses.
(A) Short-term plasticity of a train of five excitatory postsynaptic currents (EPSCs) evoked at 5 Hz for a single neuron (left, single responses to 10 trains and mean, normalised to the amplitude of …
Figure 9 with 3 supplements
Quantal analysis in autaptic neurons.
(A) Background subtracted fluorescence time series from one region of interest (ROI) from an iGluSnFR-expressing neuron at different calcium concentrations. Nominal quantal levels are indicated by …
Figure 9—figure supplement 1
Quantal nature of indicator responses.
(A) Fluorescence time series at three calcium concentrations from three regions of interest (ROIs) in an autapse expressing iGluSnFR. Quantal analysis for ROI 3 is shown in Figure 8. Note the …
Figure 9—figure supplement 2
Workflow for peak response extraction and analysis of fluorescent report of glutamate release from synapses.
A script in imageJ is used to detect regions of interest (ROIs) with changes in fluorescence over the course of movies. This determination exploits information about the number of expected responses …
Figure 9—figure supplement 3
Examples of imperfect fits to peak response histograms.
(A) For a binomial model, setting the number of releasable vesicles (N) to a too small value fails to describe the full width of the amplitude distribution at 4 mM Ca2+. At 4 mM Ca2+, the release …
Figure 10
Factors affecting the responses of iGluSnFR and SnFR-γ2.
(A) Three scenarios following glutamate release from vesicles (blue). iGluSnFR is not localised to the postsynaptic density (PSD, magenta) opposite release sites, where AMPA receptors are docked …
Videos
Video 1
Spontaneous responses of iGluSnFR expressed in bulk culture.
Video 2
Spontaneous responses of SnFR-γ2 expressed in bulk culture.
Video 3
Evoked responses of iGluSnFR expressed in autaptic neuron culture.
Video 4
Evoked responses of SnFR-γ2 expressed in autaptic neuron culture.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/84029/elife-84029-mdarchecklist1-v2.pdf
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Source code 1
imageJ macro to find regions of interest.
- https://cdn.elifesciences.org/articles/84029/elife-84029-code1-v2.zip
