Most neurons transmit information digitally using spikes that trigger the release of synaptic vesicles with low probability. The first stages of vision and hearing are distinct in that they operate with analog signals, but it is unclear how these are recoded for synaptic transmission. By imaging the release of glutamate in live zebrafish, we demonstrate that ribbon synapses of retinal bipolar cells encode contrast through changes in both the frequency and amplitude of release events. Higher contrasts caused multiple vesicles to be released within an event, and such coding by amplitude often continued after the rate code had reached a maximum frequency. Glutamate packets equivalent to five vesicles transmitted four times as many bits of information per vesicle compared with those released individually. By discretizing analog signals into sequences of numbers up to about 11, ribbon synapses can increase the dynamic range, temporal precision and efficiency with which visual information is transmitted.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
The code used to analyze the data in this study is available at https://github.com/lagnadoLab/glueSniffer.
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The authors express many thanks to J. Johnston for discussions and to H. Smulders and N. Bashford for looking after the zebrafish. Thanks are also given to M. Meyer for the gift of the constructs. This work was supported by grants to L.L. from the Wellcome Trust (102905/Z/13/Z) and an EU International Training Network (H2020-MSCA-ITN-2015-674901).
Integrated supplementary information
a) A linescan showing changes in iGluSnFR fluorescence in a synaptic terminal in response to a full-field stimulus modulated at 5 Hz (sine wave). The left-hand side shows the profile of two active zones of a terminal stimulated at 30% contrast and the right-hand side the profile of the same terminal stimulated at 100% contrast. This is one of 187 independent experiments in which the analysis procedure was applied. b) Traces extracted from the linescans shown in a. c) Expansion of the period shown boxed in b. Note that at 30% contrast there are events at one active zone that do not coincide with events at the other (highlighted by arrowheads), whereas 100% contrast drives responses in both active zones reliably over all cycles of the 5 Hz stimulus. Analysis shown in d and e were extracted from the boxed area. d) Deconvolved trace using the estimated Wiener filter. e) Estimation of the number of quanta per event. The stimulation protocol is represented below.
The temporal average (black trace) is fit with the sum of two Gaussians (red trace). Dashed blue line shows individual components. The FWHM values were 1.1 μm (left) and 0.96 μm (right).
Representative example of raw events overlayed with a Wiener kernel given by equation 6. Note that the time decay constant of the fluorescence signal is approximately 50 ms. A total number of 101 raw events were averaged for estimating the Wiener kernel filter.
a. The kymograph (top) shows the intensity profile along a line through a single terminal. The red and black traces (middle) show the time-course of the iGluSnFR signal over the two active zones marked to the right of the kymograph and the stimulus is shown immediately below. After 2 s there was a switch from constant illumination to full field modulation at 20% contrast, 5 Hz. The signals were demixed using a weighted sum of two Gaussians fit to the intensity profile along the line-scan, as described above. Two sections of the record (green boxes) are expanded in Supplementary Fig. 5. Note variations in the amplitude of glutamate transients. This example is one of 187 independent experiments in which this analysis was applied. b. The results of Wiener deconvolution applied to the traces in a using the kernel shown in Supplementary Fig. 3. The dashed red line shows an event in active zone 1 that did not coincide with an event in active zone 2, and the dashed black lines highlight events in active zone 2. c. The distribution of values in the traces shown in b (active zone 1 in red to the top and active zone 2 below in black) together with a fitted Gaussian (blue). The threshold of 3 sd above the baseline is indicated by the dashed blue arrow.
a. Activity in active zone 1 (red) and active zone 2 (black), with deconvolved traces immediately below the iGluSnFR signals. The period of activity corresponds to the large green box in Supplementary Fig. 4a. Thresholds for differentiating events from noise are shown by dashed blue lines, and the dashed vertical lines link the event in the deconvolved trace to the iGluSnFR trace. Note the burst of one large and then three smaller events around 43 s in active zone 1. Note also the deviation in the iGluSnFR signal from active zone 2 at 43 s which was not counted as event because it was small and slowly rising. This example is one of 187 independent experiments in which this analysis was applied. b. Activity in active zone 2 over the period shown by the small green box in Supplementary Fig. 4a. Note that of the three small upward deviations in the iGluSnFR signal after 29.2 s, only the second was counted as an event. c. A histogram of event amplitudes from active zone 1. The black trace is a fitted sum of six Gaussians.
Representative raw trace in response to a 5 Hz sinusoidal stimulus. The SNR value calculated for this terminal was 3.95. Top: Representative recording of the raw trace. Note that red arrows show uniquantal events. Bottom: Distribution of fluorescence values from the above recording. The red line indicates the Gaussian fit with mean 0.04, and amplitude 0.74. The black line shows the experimental data. The SNR is calculated by dividing the mean uniquantal amplitude (ΔF/F = 0.8, highlighted by gray line) by the standard deviation of the noise (0.2), represented by the Gaussian. This example is one of 187 independent experiments in which this analysis was applied.
Left: Two uniquantal events separated by 10 ms. Note that at this IEI, these events are incorrectly classified as a single 2-quanta event (bottom). Middle: Two events separated by 12 ms generate two distinguishable maxima in the deconvolved trace counted as two distinct events, the amplitude and timing of which is shown by the vertical red bars (bottom). Right: Two uniquantal events separated by 15 ms are distinguished relatively easily. In all these simulations, the SNR was 4.05. This example is one of 20 independent simulations in which similar results were observed.
These simulations suggest that in low noise conditions (SNR > 5) we can reliably distinguish events separated in time by 10–15 ms. The mean temporal discrimination window value is 12.6 ms.
a. Expression of iGluSnFR on two nearby bipolar cells b. After laser ablation of the soma of cell 1, the terminal does not show responses to a stimulus of high contrast (100%, 5 Hz), while the responses in the terminal of cell 2 remain. Scale bar: 5 µm. This example is one of 187 independent experiments in which this analysis was applied. This example is one of 4 independent experiments in which similar results were observed.
a. Confocal image of a bipolar cell expressing iGuSnFR (green) and synaptic ribbons labelled with a ribeye a antibody (red). Note the ribbons scattered throughout the inner plexiform layer. This example is one of 27 terminals in which ribbons could be counted. Scale bar 5 μm. b. In all three panels, the green image is a maximum intensity projection of the iGluSFR signal through a volume of 34 μm3 containing the terminal of the cell shown in a. A threshold was then applied to mask out fluorescence beyond the terminal. Superimposed in the top panel is a maximum intensity projection of the red signal through three planes (each separated by 0.25 μm) centred towards one side of the terminal. Two ribbons can be seen. The middle panel shows a similar projection of the red signal at a z distance of 3 μm from the first, where three ribbons can be seen. In the bottom panel, the red channel shows a maximum intensity projection through the entire volume of the terminal. Scale bar 1 μm (top panel).
Supplementary Figure 11 Model for calculating the probability of conflating signals from two ribbons.
a) Example sphere (grey line) and ellipsoid volumes (black line). The spherical cap of their intersection is defined from the radius of the sphere m (using the average nearest distance between ribbons), the circle radius at the z-value of the two volumes intersection a, and the height of the cap h. b) Top: The probability of collapse as a function of m (black line) overlayed with the values computed from the data (black circles). The mean probability is 8% (red dashed line). Bottom), the distribution of distances between ribbons. Red dashed line indicates mean nearest distance of 0.96 μm.
iGluSnFR activity at bipolar cell synapses. Imaging bipolar cell terminals expressing iGluSnFR. Video begins with 5 s constant illumination followed by modulation at 1 Hz 100% contrast (5 s), 3 Hz (5 s), 5 Hz (5 s) and 10 Hz (5 s), ending with 5 s constant illumination. Field of view: 73 µm, framerate 10 Hz. Qualitatively similar responses were observed in 187 independent experiments.
Glutamate release imaged in individual synaptic terminals. A linescan across a terminal showing changes in iGluSnFr fluorescence. Each frame shows a 100 ms interval (100 scans of the line with time proceeding upwards). After 10 s there was a switch from constant uniform illumination to modulation at 100% contrast (sine wave). There are two sources of glutamate and at ~6 s, spontaneous events can be seen in the left-hand active zone. Real time. Similar iGluSFR signals were observed in 187 independent experiments.