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.

Rearing of zebrafish. Male and female zebrafish (Danio rerio; 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).  

Isolation of zebrafish retinal RBCs. Dissociation of RBCs was performed using established procedures ⁹⁵. 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 CaCl₂, 1 mM MgCl₂, 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.

Ribeye binding peptides. 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 (>95% purity, LifeTein LLC, NJ).

Ca²⁺ indicators.

Free Ca²⁺ indicators. The potassium salts of high and low-affinity Ca²⁺ indicators Cal-520^® (high affinity, K_D 320 nM) and Cal-520N™ AM (low affinity, K_D 90 µM), referred to as Cal520HA and Cal520LA, respectively, were purchased from AAT Bioquest.

Direct conjugation of Ca²⁺ indicator to cysteine-containing ribbon-binding peptides. To target Ca²⁺ indicators to the ribbon, custom-made cysteine-containing ribeye binding peptides NH₂ -CIEDEEKPVDLTAGRRAC-COOH were synthesized and purchased from LifeTein to directly fuse with the fluorogenic 520^® maleimide (purchased from AAT Bioquest) for high affinity (HA) and low affinity (LA Ca²⁺ indicator dyes). Each peptide at one mM concentration was mixed and incubated with two mM Cal-520^® maleimide (20 mM stock solution in DMSO purchased from AAT Bioquest) for one hour at room temperature, then overnight at 4^oC. Calibration of Ca²⁺ indicator dyes was performed as described ²⁵ and as detailed below. The conjugated Ca²⁺ indicators were stored at -20°C in smaller aliquots, each sufficient for a single day's experiments.

Measurement of dissociation constants (K_D) for Ca²⁺ indicator peptides. The effective *K_D *(K_eff) was obtained by measuring the fluorescence of Cal520HA-RBP and TAMRA-RBP in buffered Ca²⁺ solutions, determining the ratio between them, and using the Grynkiewicz equation to determine the Ca²⁺ concentration [Ca^2+^] from this ratio. However, this was not possible for the low-affinity indicator Cal520LA due to the large Ca²⁺ levels required to calibrate it. Thus, K_eff for Cal520LA-RBP could be larger than the~ _K1/2 provided by the manufacturer for Cal520LA (~, 90 mM), as reported previously with K, ~measurements of OB-5N in inner hair cells ⁴². Thus, our estimates of local Ca²⁺ 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 K_eff estimate, due to potential dye saturation effects.

RBC voltage clamp recording. 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 MgCl₂, 0.2 mM* N*-methyl-d-glucamine-EGTA, 2 mM Na₂ATP, 0.5 mM Na₂GTP, 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 Ca²⁺ indicators Cal520HA-free (Figs. 1 and 2) and Cal520LA-free (Fig. 2, 3 and 5) to demonstrate our nanophysiological approach or 2) ribeye-bound Ca²⁺ indicators Cal520HA-RBP (Figs. 3  and Supplementary Figs. 3 and 5) and Cal520LA-RBP (Figs. 3, 4, 5, and 8) to measure local ribbon-associated Ca²⁺ signals. Current responses from the cell membrane were recorded under a voltage clamp with a holding potential (V_H) of -65 mV that was stepped to 0 mV (t₀) 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 .

Acquisition of confocal images. 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) ³² and Ca²⁺ 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 (Fig.1Aii) and to monitor local changes in Ca²⁺ concentration at a single ribbon, as we demonstrated previously, to estimate the Ca²⁺ 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 (Fig. 1Ai) 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 Ca²⁺ concentration along the ribbon axis (Fig. 1Aii). 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 Ca²⁺ signals (Fig.1C, cyan) 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 (Fig.1B). 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 Ca²⁺ 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 Ca²⁺ indicators, and line-scan images were collected and analyzed using the same procedures used for experimental samples.

Point spread function. 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.

Photobleaching. 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.

Data analysis. 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.