High-speed imaging of glutamate release with genetically encoded sensors

Abstract

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

Fig 1. Overview of the protocol workflow for the development of glutamate sensors and 2-photon imaging of glutamate transients in individual synapses.

Fig 2. Characterization of GEGIs.

(a) Left: Setup for affinity and selectivity determination. GEGI in assay buffer is placed into a fluorescence cuvette with a magnetic stirrer and placed inside the sample chamber of the fluorescence spectrometer (Fluorolog3, Horiba Scientific). With an Aladdin pump, the ligand (Glu, Asp, Ser) is continuously added to the cuvette while the fluorescence is recorded (λ_ex = 492 nm, λ_ex = 514 nm). Right: Examples of affinity curves of a GEGI for glutamate (black squares) and aspartate (black circles). The fluorescence emission is corrected for dilution and bleaching and plotted against the glutamate concentration in the chamber. The data is then fitted with a Hill equation (green and orange traces for glutamate and aspartate, respectively). (b) Left: Setup for stopped-flow kinetic measurement. The solutions are rapidly mixed in the mixing chamber and then pushed into the optical cell where the fluorescence is excited at 492 nm and emission is detected by a PMT with a cut-off filter (>530 nm). For association the GEGI in assay buffer without glutamate is loaded into the drive syringe B and mixed with assay buffer containing increasing concentrations of glutamate filled in drive syringe A. For dissociation the GEGI in assay buffer with saturating glutamate concentration is loaded into the drive syringe B and mixed with GluBP 600n in assay buffer filled into drive syringe A. For both measurements the PMT zero level is determined by mixing assay buffer with assay buffer and the intrinsic fluorescence of the GEGI is recorded by mixing the GEGI in assay buffer with assay buffer (both without Glu). Right: Examples for recorded time traces. Top: Fluorescence increase observed when GEGIs are mixed with increasing glutamate concentration. Bottom: Decrease in fluorescence when glutamate is retained from GEGI by GluBP 600n. The raw data are fitted with monoexponential decays (dark green line).

Fig 3. iGluSnFR expression in CA3 pyramidal cells in organotypic slice culture of rat hippocampus.

(a) Co-expression of two plasmids in individual CA3 pyramidal cells in organotypic slice culture. The red fluorescent protein tdimer2 labels the axoplasm while the membrane-anchored iGluSnFR is exposed to the extracellular space. (b) Transmitted light image (dark field) of a transfected organotypic culture merged with a wide-field fluorescence image showing three transfected CA3 neurons. The area for synaptic imaging is indicated (red dotted box). Scale bar represents 500 μm. (c) Two-photon image stack (maximum intensity projection) of CA3 axons in CA1 stratum radiatum (cells not identical to panel b). Scale bar represents 10 μm. Image from . (d) Maximum intensity projection of two-photon images of CA3 pyramidal neuron expressing iGlu_u 4 days after electroporation (fluorescence intensity is shown as inverted gray values). iGlu_u shown here and other GEGIs had their fluorescence mainly localized to the plasma membrane over the entire cell. The scale bar represents 50 μm (left image) and 5 μm (right image). (e) Action potentials are elicited in a transfected neuron by somatic current injections and glutamate release is simultaneously optically recorded (GEGI fluorescence) from a single Schaffer collateral bouton in CA1, showing a broad distribution of amplitudes and occasional failures. Images were acquired at 500 Hz at 34°C. Individual trials are classified as successes if the peak amplitude of the GEGI transient is >2σ (green traces) and as failures when the peak amplitude is <2σ (gray traces). Note propagation delay between presynaptic APs and glutamate release events at distal bouton.

Fig 4. Localization of fluorescence transients in low and high [Ca²⁺]_o.

(a) Morphology of individual boutons. Red fluorescence was upsampled (16 x 16 pixels to 128 x 128 pixels), aligned and averaged over all trials. Scale bars represent 0.5 μm. (b) Average response of iGluSnFR superimposed with bouton outline (black line) from red channel (morphology). The bouton outline was generated by thresholding the red channel followed by smoothing. (c) Two-dimensional Gaussian fit to average response. On average, the full width at half maximum (FWHM) was 763 ± 29 nm (n = 12; 5 boutons shown here) (d) Plotting the center position of 2D Gaussian fits to individual trials. Fusion appears to be localized to a small region on the bouton (active zone). Amplitude (ΔF/F₀) of individual trials is color-coded. Scale bars represent 0.5 μm. (e) Increasing the extracellular Ca²⁺ concentration increased the amplitude of individual responses, but did not lead to release events outside the active zone. (f) Fitting responses classified as failures (< 2σ of baseline noise) did not reveal any clustering, indicating that there was indeed no localized signal in these trials (true negatives). (g) Fitting frames before stimulation (green baseline fluorescence) did also not result in clustering.

Fig. 5. Signal extraction of GEGI transients from a single Schaffer collateral bouton in CA1.

(a) The spatial extent of iGluSnFR fluorescence transients was 760 nm, on average (FWHM, short axis of Gaussian fits). No deconvolution was applied. (b) Sampling the surface of the bouton by traditional raster scanning requires extreme acceleration of the scan mirrors at the turning points, leading to large positional errors. Spiral scans avoid sharp direction changes (no flyback) and can, therefore, sample the entire bouton surface in 1 or 2 ms. Due to the elongated PSF (1.8 μm in the axial direction), upper and lower surface of a bouton are sampled simultaneously. (c) Plotting the unfolded spiral scan lines vs. time (single trial). Raw fluorescence intensity is coded in pseudocolors. At t = 58 ms, a glutamate release event from an individual presynaptic terminal occurred and was sampled twice during every spiral scan. (d) Only columns with ΔF > ½ max (ΔF) were analyzed (ROI, region of interest). Green trace: Extracted fluorescence transient (before bleach correction). (e) Upper panel: Average of 10 trials (single APs) to analyze lateral spread of signal from t=0 to t=18 ms. Lower panel: Decay of fluorescence transient (5 scan lines plotted = 18 ms). Note the lack of lateral spread of the signal due to slow diffusion of membrane-anchored GEGI. (f) iGluSnFR response amplitude (green markers) of a single bouton stimulated with single APs every 10 s. Note that response amplitudes were constant over time. A time window before stimulation was analyzed to estimate imaging noise (gray markers). The histogram of response amplitudes shows separation between failures of glutamate release (overlap with the baseline histogram) and successes.

Fig. 6. Release statistics of neighboring boutons on the same axon.

(a) Glutamate transients (green dots) and baseline fluorescence (grey dots) of two neighboring boutons measured in ACSF containing 2 mM Ca²⁺ and 1 mM Mg²⁺ located on the same axon (left panels) and their corresponding histogram counts (right panels). (b) Glutamate transients (blue dots) and baseline fluorescence (grey dots) of two neighboring boutons located on the same axon (left panels) and their corresponding histograms (right panels) measured in ACSF containing 2 mM Ca²⁺ and 1 mM Mg²⁺ and their corresponding histogram counts (right panels). (c) Synaptic release probability (pr) (calculated out of ~ 100 trials) of individual boutons (B1) and their neighboring bouton on the same axon (B2); n=10. The pair of neighboring boutons from (a) and (b) are shown in green and blue, respectively. (d) Histogram of Δ p_r = |p_r BX-p_r BY|. BX and BY are randomly paired from the dataset in (c). |Δp_r B2- Δp_r B1| (red vertical line) is significantly more similar than mean Δ p_r of two boutons paired randomly from the same dataset; (p-value: 0.0148). (e) Amplitude of the iGluSnFR signal given a success of a bouton B1 and its neighbor on the same axon (B2); n=10. The pair of neighboring boutons from (a) and (b) are shown in green and blue, respectively. (f) Histogram count of the difference between the average ΔF/F₀ of successes only of two random neighboring boutons. The difference of the average ΔF/F₀ of successes from two neighboring boutons (red vertical line) is not significantly more similar than the randomly connected pairs of boutons.

Fig. 7. Resolving high-frequency transmission with ultrafast GEGI, iGl_u.

(a) The presynaptic neuron was driven to spike at 100 Hz (10 APs). After a pause of 0.5 s, one more AP was triggered to quantify recovery from depression. iGluSnFR signals (blue) or iGlu_u signals (green) were recorded at single Schaffer collateral boutons (only during the 100 Hz train) in stratum radiatum. Recordings were performed in 4 mM Ca²⁺ and 1 mM Mg²⁺ to ensure very high release probability. Note summation and saturation of iGluSnFR (but not iGlu_u) during the high-frequency train. (b) iGlu_u responses to the 1^st AP of the 100 Hz train, to the 9^th AP of the train, and to the recovery pulse. To minimize bleaching, the bouton was only imaged (spiral scans) during pulses 1, 9 and 11. Note frequent failures in response to pulse 9. (c) Extracted single-trial amplitudes reveal strong depression and full recovery of this bouton. Failures of glutamate release can be seen in response to pulse 9. Note the large amplitude of initial responses compared to depressed responses. Plots were generated with violinplot.m (GitHub, ©Bastian Bechtold).