DOI: 10.5061/dryad.qfttdz0vh
title: Data from: Nanophysiology approach reveals diversity in calcium microdomains across Zebrafish retinal bipolar ribbon synapses

description: Rapid and high local calcium (Ca2+) signals are essential for triggering
 neurotransmitter release from presynaptic terminals. In specialized
 bipolar ribbon synapses of the retina, these local Ca2+ signals control
 multiple processes, including the priming, docking, and translocation of
 vesicles on the ribbon before exocytosis, endocytosis, and the
 replenishment of release-ready vesicles to the fusion sites for sustained
 neurotransmission. However, our knowledge about Ca2+ signals along the
 axis of the ribbon active zone is limited. Here, we used fast confocal
 quantitative dual-color ratiometric line-scan imaging of a fluorescently
 labeled ribbon binding peptide and Ca2+ indicators to monitor the spatial
 and temporal aspects of Ca2+ transients of individual ribbon active zones
 in zebrafish retinal rod bipolar cells (RBCs). We observed that a Ca2+
 transient elicited a much greater fluorescence amplitude when the Ca2+
 indicator was conjugated to a ribeye-binding peptide than when using a
 soluble Ca2+ indicator, and the estimated Ca2+ levels at the ribbon active
 zone exceeded 26 μM in response to a 10-millisecond stimulus, as measured
 by a ribbon-bound low-affinity Ca2+ indicator. Our quantitative modeling
 of Ca2+ diffusion and buffering is consistent with this estimate and
 provides a detailed view of the spatiotemporal [Ca2+] dynamics near the
 ribbon. Importantly, our data demonstrates that the local Ca2+ levels may
 vary between ribbons of different RBCs and within the same cells. The
 variation in local Ca2+ signals is found to correlate with ribbon size and
 active zone extent. Our serial electron microscopy results provide new
 information about the heterogeneity in ribbon size, shape, and area of the
 ribbon in contact with the plasma membrane.
description: <strong>Rearing of zebrafish.</strong> Male and
 female zebrafish (<em>Danio rerio</em>; 16 ~20 months) were
 raised under a 14 h light/10 h dark cycle and housed according to NIH
 guidelines and the University of Tennessee Health Science Center (UTHSC)
 Guidelines for Animals in Research. All procedures were approved by the
 UTHSC Institutional Animal Care and Use Committee (IACUC; protocol #
 23-0459).   <strong>Isolation of zebrafish
 retinal RBCs.</strong> Dissociation of RBCs was performed using
 established procedures <sup>95</sup>. Briefly, retinas were
 dissected from zebrafish eyes and incubated in hyaluronidase (1100
 units/ml) for 20 minutes. The tissue was washed with a saline solution
 containing 120 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl<sub>2</sub>, 1
 mM MgCl<sub>2</sub>, 10 mM glucose, and 10 mM HEPES, pH = 7.4]
 before being cut into quadrants. Each quadrant was incubated at room
 temperature for 25 to 40 min in the same saline solution, to which was
 added DL-cysteine and papain (20-30 units/ml; Sigma Millipore, St. Louis,
 MO) and triturated using a fire-polished glass Pasteur pipette. Individual
 cells were transferred to glass-bottomed dishes, allowed to attach for 30
 minutes, and washed with saline solution before being used for
 experiments. <strong>Ribeye binding
 peptides</strong>. As a means of localizing the ribbons, custom
 peptides containing the ribbon binding sequence fused to TAMRA
 (tetramethylrhodamine; TAMRA: GIDEEKPVDLTAGRRAG) dye were synthesized,
 purified, and purchased from LifeTein (&gt;95% purity, LifeTein LLC,
 NJ).
 <strong>Ca<sup>2+</sup></strong>
 <strong>indicators.</strong> <em>Free
 Ca<sup>2+</sup></em> <em>indicators</em>.
 The potassium salts of high and low-affinity Ca<sup>2+</sup>
 indicators Cal-520<sup>®</sup> (high affinity,
 <em>K<sub>D</sub></em> 320 nM) and Cal-520N™ AM
 (low affinity, <em>K<sub>D</sub></em> 90 µM),
 referred to as Cal520HA and Cal520LA, respectively, were purchased from
 AAT Bioquest. <em>Direct conjugation of
 Ca<sup>2+</sup></em> indicator to cysteine-containing
 ribbon-binding peptides. To target Ca<sup>2+</sup> indicators
 to the ribbon, custom-made cysteine-containing ribeye binding peptides
 NH<sub>2</sub> -CIEDEEKPVDLTAGRRAC-COOH were synthesized and
 purchased from LifeTein to directly fuse with the fluorogenic
 520<sup>®</sup> maleimide (purchased from AAT Bioquest) for
 high affinity (HA) and low affinity (LA Ca<sup>2+</sup>
 indicator dyes). Each peptide at one mM concentration was mixed and
 incubated with two mM Cal-520<sup>®</sup> maleimide (20 mM
 stock solution in DMSO purchased from AAT Bioquest) for one hour at room
 temperature, then overnight at 4<sup>o</sup>C. Calibration of
 Ca<sup>2+</sup> indicator dyes was performed as described
 <sup>25</sup> and as detailed below. The conjugated Ca²⁺
 indicators were stored at -20°C in smaller aliquots, each sufficient for a
 single day's experiments.
 <strong>Measurement of dissociation constants
 (K<sub>D</sub>) for Ca<sup>2+</sup></strong>
 <strong>indicator peptides</strong>. The
 effective *K<sub>D</sub> *(<em>K</em><sub>eff</sub>) was obtained by measuring the fluorescence of Cal520HA-RBP and TAMRA-RBP in buffered Ca<sup>2+</sup> solutions, determining the ratio between them, and using the Grynkiewicz equation to determine the Ca<sup>2+</sup> concentration [Ca^2+^] from this ratio. However, this was not possible for the low-affinity indicator Cal520LA due to the large Ca<sup>2+</sup> levels required to calibrate it. Thus, <em>K</em><sub>eff</sub> for Cal520LA-RBP could be larger than the~ <em><sub>K1/2</sub></em> provided by the manufacturer for Cal520LA (~, 90 mM), as reported previously with <em>K</em>, ~measurements of OB-5N in inner hair cells <sup>42</sup>. Thus, our estimates of local Ca<sup>2+</sup> concentrations obtained using Cal520LA represent the lower bounds of the underlying true values. As noted in the Results section, the same may be true for the high-affinity Cal520HA despite its accurate <em>K</em><sub>eff</sub> estimate, due to potential dye saturation effects. <strong>RBC voltage clamp recording.</strong> Whole-cell patch-clamp recordings were made from isolated RBCs, as described previously. Briefly, a patch pipette containing pipette solution (120 mM Cs-gluconate, 10 mM tetraethyl-ammonium-Cl, 3 mM MgCl<sub>2</sub>, 0.2 mM* N*-methyl-d-glucamine-EGTA, 2 mM Na<sub>2</sub>ATP, 0.5 mM Na<sub>2</sub>GTP, 20 mM HEPES, pH = 7.4) was placed on the synaptic terminal, as described previously. The patch pipette solution also contained a fluorescently-labeled RBP peptide (TAMRA-RBP) to mark the positions of the ribbons and either 1) free Ca<sup>2+</sup> indicators Cal520HA-free (<strong>Figs. 1 and 2</strong>) and Cal520LA-free (<strong>Fig. 2, 3 and 5</strong>) to demonstrate our nanophysiological approach or 2) ribeye-bound Ca<sup>2+</sup> indicators Cal520HA-RBP (<strong>Figs. 3  and Supplementary Figs. 3 and 5</strong>) and Cal520LA-RBP (<strong>Figs. 3, 4, 5, and 8</strong>) to measure local ribbon-associated Ca<sup>2+</sup> signals. Current responses from the cell membrane were recorded under a voltage clamp with a holding potential (V<sub>H</sub>) of -65 mV that was stepped to 0 mV (t<sub>0</sub>) for 10 milliseconds. These responses were recorded with a patch clamp amplifier running PatchMaster software (version v2x90.4; HEKA Instruments, Inc., Holliston, MA). Membrane capacitance, series conductance, and membrane conductance were measured via the sine DC lock-in extension in PatchMaster and a 1600 Hz sinusoidal stimulus with a peak-to-peak amplitude of 10 mV centered on the holding potential . <strong>Acquisition of confocal images.</strong> Confocal images were acquired using an Olympus model IX 83 motorized inverted FV3000RS laser-scanning confocal microscopy system (Olympus, Shinjuku, Tokyo, Japan) running FluoView FV31S-SW software (Version 2.3.1.163; Olympus, Center Valley, PA) equipped with a 60 X silicon objective (NA 1.3), all diode laser combiner with five laser lines (405, 488, 515, 561 & 640 nm), a true spectral detection system, a hybrid galvanometer, and a resonant scanning unit. Fluorescently labeled ribeye binding peptide (RBP) <sup>32</sup> and Ca<sup>2+</sup> indicator were delivered to RBC via a whole-cell patch pipette placed directly at the cell terminal. We waited for 30 s after break-in to allow Cal520HA to reach equilibrium with the patch-pipette before obtaining the first fluorescence image.  Rapid x-t line scans at the ribbon location were performed to localize synaptic ribbons (<strong>Fig.1Aii</strong>) and to monitor local changes in Ca<sup>2+</sup> concentration at a single ribbon, as we demonstrated previously, to estimate the Ca<sup>2+</sup> levels at the plasma membrane and to track a single synaptic vesicle at ribbon locations.  The z-projection from a series of confocal optical sections through the synaptic terminal (<strong>Fig. 1Ai</strong>) illustrates ribbon labeling (magenta spots). RBP fluorescence was used to localize a synaptic ribbon and to define a region for placing a scan line perpendicular to the plasma membrane, extending from the extracellular space to the cytoplasmic region beyond the ribbon to monitor changes in the Ca<sup>2+</sup> concentration along the ribbon axis (<strong>Fig. 1Aii</strong>). The focal plane of the TAMRA-RBP signal was carefully adjusted for sharp focus to avoid potential errors arising from the high curvature near the top of the terminal and the plane of membrane adherence to the glass coverslip at the bottom of the terminal. Sequential dual laser scanning was performed at rates of 1.51 milliseconds per line. Two-color laser scanning methods allowed observation of Ca<sup>2+</sup> signals (<strong>Fig.1C, cyan</strong>) throughout the full extent of the ribbon in voltage-clamped synaptic terminals, while the ribbon and cell border were imaged with a second fluorescent label. The Exchange of TTL (transistor-transistor logic) pulses between the patch-clamp and imaging computers synchronized the acquisition of electrophysiological and imaging data. The precise timing of imaging relative to voltage-clamp stimuli was established using PatchMaster software to digitize horizontal-scan synced pulses from the imaging computer in parallel with the electrophysiological data (<strong>Fig.1B</strong>). Acquisition parameters, such as pinhole diameter, laser power, PMT gain, scan speed, optical zoom, offset, and step size were kept constant between experiments. Sequential line scans were acquired at 1-2 millisecond/line and 10 ms/pixel with a scan size of 256 × 256 pixels. Bleed-through between the channels was confirmed with both lasers using the imaging parameters we typically use for experiments. To test bleed-through from the RBP channel (TAMRA-RBP) to the Ca<sup>2+</sup> indicator (Cal-520HA and Cal-520LA), whole-cell recordings from RBC terminals were performed with patch pipette solution that contained TAMRA-RBP or the aforementioned Ca<sup>2+</sup> indicators, and line-scan images were collected and analyzed using the same procedures used for experimental samples. <strong>Point spread function.</strong> The lateral and axial point spread function (PSF) was obtained as described previously. Briefly, an XYZ scan was performed through a single 27 nm bead and the maximum projection in the xy-plane was fit to the Gaussian function using Igor Pro software. We obtained the full width at half maximum (FWHM) values for x and y-width of 268 and 273 nm, respectively, in the lateral (x-y plane) and for y-z width of 561 nm in the axial (y-z axis) resolution. <strong>Photobleaching.</strong> We minimized photobleaching and phototoxicity during live-cell scanning by using fast scan speed (10 microsec/pixel), low laser intensity (0.01–0.06% of maximum), and low pixel density (frame size, 256 × 256 pixels). We estimated photobleaching using x-t line scans of Cal520HA or Cal520LA and TAMRA-RBP in the absence of stimulation, with the same imaging parameters used for experimental samples. <strong>Data analysis.</strong> Quantitative FluoView x-t and x-y scans were analyzed initially with ImageJ software (imagej.nih.gov) and subsequently with Igor Pro software (Wavemetrics, Portland OR) for curve fitting and production of the figures. Data from PatchMaster software were initially exported to Microsoft Excel (Version 16.81) for normalizing and averaging and exported from MS Excel to Igor Pro (Version 9.05) for curve fitting and production of the l figures.
description: # Data from: Nanophysiology approach reveals diversity in calcium
 microdomains across Zebrafish retinal bipolar ribbon synapses Dataset DOI:
 [10.5061/dryad.qfttdz0vh](https://doi.org/10.5061/dryad.qfttdz0vh) ##
 Description of the data and file structure The present dataset compares
 calcium domains in retinal rod bipolar cells using free and ribeye-bound
 calcium indicators. Computational models were established to estimate the
 spatial distribution of the Ca2+ signals accurately. The ultrastructure of
 the zebrafish retina, obtained using Serial Block-Face Scanning Electron
 Microscopy, was used to count and measure the rod bipolar cell synaptic
 ribbons and active zones. Experiment data and quantities contained in the
 spreadsheets are described below under `Description` and `Glossary`
 sections. For a detailed description of the experiments outlined here, see
 the associated article at
 [https://elifesciences.org/reviewed-preprints/105875](https://elifesciences.org/reviewed-preprints/105875). ### Files and variables #### File: 3._eLife_source_data.xlsx **Description:**  Fig 1D: Fluorescence intensity profiles along the x-axis for RBP (magenta) and Cal520HA before stimuli (gray line), during 10 ms depolarization (cyan line), and after stimuli (black line) depolarization were obtained by averaging three pixels along the time-axis. Fig. 1F: Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal (light cyan), distal (dark cyan), and cytoplasmic (gray) locations from the single ribbon. Fig.2A: Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal (light cyan) and distal (dark cyan) locations from a single ribbon. Fig.2B: Spatially averaged Cal520LA fluorescence as a function of time at ribbon proximal (light cyan) and distal (dark cyan) locations. Fig.3C: Spatially averaged fluorescence of Cal520HA-RBP as a function of time at ribbon proximal (light cyan), and distal (dark cyan) locations. Fig.3D: Spatially averaged fluorescence of Cal520LA-RBP as a function of time at ribbon proximal (light cyan), and distal (dark cyan) locations. Fig.3E: Average fluorescence intensity of proximal Ca^2+^ signals obtained with Cal520LA-RBP (light cyan and dark cyan, respectively) and Cal520LA-free (grey). Fig.3F: Average fluorescence intensity of distal Ca^2+^ signals obtained with Cal520LA-RBP (light cyan and dark cyan, respectively) and Cal520LA-free (grey). Fig.4C: Spatially averaged Cal520LA-RBP as a function of time at ribbon proximal (light cyan), distal (dark cyan), and non-ribbon locations (blue). Fig.4D: Ca^2+^ measurements along the ribbon axis using the nanophysiology approach. Fig.5A: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan) and distal (dark cyan) Ca^2+^ signals with Cal520LA-RBP as a function of time at distinct ribbon locations with pipette solution containing 0.2 mM EGTA. Fig.5B: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan) and distal (dark cyan) Ca^2+^ signals with Cal520LA-RBP as a function of time at distinct ribbon locations with pipette solution containing 2 mM EGTA. Fig.5C: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan) and distal (dark cyan) Ca^2+^ signals with Cal520LA-RBP as a function of time at distinct ribbon locations with pipette solution containing 10 mM EGTA. Fig.5D: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan) and distal (dark cyan) Ca^2+^ signals with Cal520LA-RBP as a function of time at distinct ribbon locations with pipette solution containing 2 mM BAPTA. Fig.5E: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal Ca^2+^ signals measured with Cal520LA-RBP (cyan) and Cal520LA-free (gray) as a function of time with pipette solution containing 2 mM BAPTA. The corresponding maximum values of trial-averaged Δ*F*/*F*~rest~ was significantly higher with 2mM Cal520LA-bound BAPTA than 2 mM Cal520LA free BA Fig.8B: Variability of proximal Ca^2+^ signals in different ribbons across different cells in the presence of 10 mM EGTA, recorded using Cal520LA-RBP. Fig.8C: Variability of distal Ca^2+^ signals in different ribbons across different cells in the presence of 10 mM EGTA, recorded using Cal520LA-RBP. Fig.8E: Comparing [Ca^2+^] across various ribbons of an individual RBC. Ribbon variability within individual cells was measured with 10 mM EGTA using Cal520LA-RBP at proximal locations. Fig.8F: Comparing [Ca^2+^] across various ribbons of an individual RBC. Ribbon variability within individual cells was measured with 10 mM EGTA using Cal520LA-RBP at distal locations. Fig.9C: Measurements obtained from Serial Block-Face Scanning Electron Microscopy to obtain the ribbon distance for the three RBCs. Fig.9D: Measurements obtained from Serial Block-Face Scanning Electron Microscopy to obtain the distances between each ribbon and its five nearest ribbons for all ribbons contained in the three RBCs. Fig.10E: Summary of the active zone size, the area of the ribbon associated with the plasma membrane measured in serial sections across the three RBCs. Fig.11B: Scatter plot of maximal Ca^2+^ influx (F/F~rest~) vs. TAMRA-RBP. Fig.11C: Scatter plot of maximal synaptic ribbon length vs. maximal synaptic ribbon width as estimated from SBF-SEM images Fig.11D: Scatter plot of area of AZ vs. ribbon width (filled black circles) and of area of AZ vs. ribbon length (filled grey circles). Fig.5 -fig Supp.3A: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan), and distal (dark cyan) Ca^2+^ signals measured with Cal520HA-RBP as a function of time for distinct ribbon locations with pipette solution containing 0.2 mM EGTA.   Fig.5 -fig Supp.3B: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan), and distal (dark cyan) Ca^2+^ signals measured with Cal520HA-RBP as a function of time for distinct ribbon locations with pipette solution containing 2 mM EGTA. Fig.5 -fig Supp.3C: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan), and distal (dark cyan) Ca^2+^ signals measured with Cal520HA-RBP as a function of time for distinct ribbon locations with pipette solution containing 10 mM EGTA. Fig.5 -fig Supp.3D: Average temporal fluorescence intensity (normalized to DF/F~rest~) of proximal (light cyan), and distal (dark cyan) Ca^2+^ signals measured with Cal520HA-RBP as a function of time for distinct ribbon locations with pipette solution containing 2 mM BAPTA. Fig.6 -fig Supp.4A: Estimation of Ca^2+^ current per ribbon.**  **The average Ca^2+^ current (dark gray) and the number of synaptic ribbon fluorescence labeled with TAMRA-RBP (light gray) suggested that retinal rod bipolar ribbon synapses have a Ca^2+^ current of 1pA/ribbon. Fig.8 -fig Supp.5A: Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal location. Fig.8 -fig Supp.5B: Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal location. Fig.10 -fig Supp.8: Summary of the area of the individual ribbon associated with the plasma membrane measured in serial sections of each of the three RBCs. Supplementary File 1: Effect of exogenous Ca^2+^ chelators alter Ca^2+^ signals gradient along synaptic ribbon measured with Cal520LA-RBP. Supplementary File 2:  Effect of exogenous Ca^2+^ chelators alters the Ca^2+^ signal gradient along the synaptic ribbon measured with Cal520H-RBP. Supplementary File 3: Model parameters for Ca^2+^ diffusion, buffering, and clearance. ##### ## Glossary \- RBP: Ribeye-binding peptide \- RBC: rod bipolar cells. \- Cal520HA: Higher-affinity free calcium indicator \- Cal520LA: Lower-affinity free calcium indicator \- Cal520HA-RBP: Higher-affinity calcium indicator fused to ribeye binding peptide \- Cal520LA-RBP: Lower-affinity calcium indicator fused to ribeye binding peptide \- TAMRA-RBP: RBP fluorescently labeled with TAMRA \- EGTA: ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid \- BAPTA: 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. ## Code/software * [https://github.com/vaithianathanlab/calcium_microdomains_project](https://github.com/vaithianathanlab/calcium_microdomains_project): R codes used for statistical analysis * [https://github.com/mvvik/Zebrafish-Calcium-Microdomains](https://urldefense.com/v3/__https:/github.com/mvvik/Zebrafish-Calcium-Microdomains__;!!DLa72PTfQgg!IEswyA9FjlfCVn3hpaR4EI7jSFrUsylmvZs5BhGCS9gJ0PctZ6mL_r-b2_EcVFjEPkM-I0keO5jyoQbng60$ "https://urldefense.com/v3/__https://github.com/mvvik/Zebrafish-Calcium-Microdomains__;!!DLa72PTfQgg!IEswyA9FjlfCVn3hpaR4EI7jSFrUsylmvZs5BhGCS9gJ0PctZ6mL_r-b2_EcVFjEPkM-I0keO5jyoQbng60$"): MATLAB & CalC code for model simulations in the manuscript ## Access information Other publicly accessible locations of the data: * Not applicable Data was derived from the following sources: * Not applicable