Nanophysiology approach reveals diversity in calcium microdomains across
- Department of Pharmacology, Addiction Science, and Toxicology, The University of Tennessee Health Science Center, United States
- Department of Neuroscience, University of Wisconsin, United States
- McPherson Eye Research Institute, University of Wisconsin, United States
- Department of Ophthalmology and Visual Sciences, University of Wisconsin, United States
- Department of Mathematical Sciences, New Jersey Institute of Technology, United States
- Department of Cellular and Molecular Physiology, Yale University School of Medicine, United States
- Department of Ophthalmology and Visual Sciences, Yale University School of Medicine, United States
- Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, United States
Figures
Figure 1 with 2 supplements
Nanophysiology approach unveils spatiotemporal properties of local Ca2+ signaling in retinal rod bipolar cells (RBC) terminals.
(A, Left panel) Single projection from a series of confocal optical sections through a zebrafish RBC synaptic terminal. A synaptic terminal was voltage-clamped using a whole-cell pipette with an internal solution containing TAMRA-RBP (magenta) to label synaptic ribbons (a, magenta). Ribbon binding peptide (RBP) fluorescence was concentrated at ribbons and also filled the entire terminal, allowing visualization of the terminal border. Experiments were carried out on ribbons that could be distinguished from adjacent ribbons (white asterisks). Scale, 5 μm. Right panel, Close-up view of a single synaptic ribbon. The outside of the cell is to the right, and x-t scan lines (dotted lines) were positioned perpendicular to the plasma membrane, extending from the intracellular side of the ribbon to the extracellular space. A rapid x-t line scan was taken at a ribbon location perpendicular to the plasma membrane across a ribbon (b) with sequential dual laser scanning performed at rates of 1.51 milliseconds per line per channel (3.02 milliseconds per line for both channels). The resulting x-t raster plots were used to measure the fluorescence intensity profiles of RBP (magenta) and the Ca2+ transient (cyan; Cal520HA) shown in panels C and E. Scale, 1.6 μm. (B) Voltage-clamp recording of a RBC terminal. Terminals were held at –65 mV and stepped to 0 mV (t0) for 10 ms (red) to evoke a brief Ca2+ current (black). A typical experiment began with a voltage command (VH = –65 mV), and a transistor-transistor logic (TTL) pulse generated by the Patch Master software (t1) triggers image acquisition (t2). t0 is the time of depolarization. (C) Illustration of the approach used to obtain the spatial location of Ca2+ signals with respect to the ribbon. Example of an x-t raster plot that is oriented to illustrate the x-axis intensity profiles of RBP (magenta) and Ca2+ signal (cyan) fluorescence during a brief depolarization. Sequential dual laser scanning was performed at 1.51 milliseconds per line for one channel (3.02 milliseconds per line for both channels). (D) 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. RBP (magenta) and Ca2+ signals during (light cyan) were fit with a Sigmoid-Gaussian function (Vaithianathan et al., 2016). The centroid (x-axis position) of the RBP (magenta) and Ca2+ signals during (cyan) were taken as the peak of the Gaussian fit (x0). The parameter x1/2 (dotted magenta line) from the Sigmoid fit to the RBP fluorescence (magenta trace) was used to estimate the location of the plasma membrane. (E) The x-t raster plot shown was from the same recording as in panel C but re-oriented to demonstrate the t-axis intensity profiles of RBP (magenta) and Ca2+ transient (cyan). (F) 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 shown in D, upper panel.
Figure 1—figure supplement 1
Ultrastructure of a zebrafish rod bipolar cell (RBC) showing distinct synaptic vesicle pools.
(A) A single section from serial block-face scanning electron microscopy (SBF-SEM) shows RBCs (purple-shaded) and postsynaptic neurons (cyan and brown-shaded) in the SEM volume. Synaptic vesicles in RBCs are distributed among at least four distinct pool types based on their fusion kinetics, the average proximity of vesicles to Cav, and the state of vesicle preparedness for Ca2+-triggered fusion. All of the vesicles in the ribbon (magenta-shaded) readily releasable pools (RRP) are molecularly prepared for fusion but differ in their anatomical distance to Cav, leading to a clear kinetic distinction between the first and second phases of exocytosis triggered by strongly activating the Ca2+ current. The RRP vesicles docked at the base of the synaptic ribbon are defined as the ultrafast releasable pool (UFRP; panel B, green vesicles) (Mennerick and Matthews, 1996), which are distinct from the remaining RRPs at the ribbon that are distal to the plasma membrane (PM; B; yellow vesicles). These anatomically distinct pools contribute to the rapid first phase and slower second phase of neurotransmitter release, respectively (Coggins and Zenisek, 2009; Mennerick and Matthews, 1996; Singer and Diamond, 2003; Neves and Lagnado, 1999; von Gersdorff and Matthews, 1997). The cytoplasmic pool that replenishes the ribbon pools is defined as the recycling pool (RP, orange vesicles), while those that do not participate in neurotransmitter release are members of a reserve pool (Res. P, blue vesicles). Scale bars: 500 nm (panels A and B). (B) The same single SBF-SEM section from A, but without shading.
Figure 1—figure supplement 2
Ca2+ indicator fluorescence imaging in the rod bipolar cell (RBC) terminal.
(A) Measurement of the point-spread function (PSF) of the microscope. (a) Image of a single 27 nm bead. Scale bar: 250 nm (b) Two-dimensional Gaussian fitted to panel A using Igor Pro software. The indicated standard deviations (SD) (114 and 116 nm) correspond to a full width at half maximum (FWHM) of 268 and 273 nm in the x and y axes, respectively. (c) Superposition of fit (blue) with bead image (red). (B) RBC isolated from zebrafish retina after papain digestion and immunostained with anti-PKCα antibodies shows its characteristic flask-shaped cell body and large terminals.
Figure 2
Kinetics of Ca2+ transients in response to brief stimuli recorded with freely diffusible indicators.
(A) Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal (light cyan) and distal (dark cyan) locations from a single ribbon, as shown in Figure 1 (n=24 ribbons from seven different rod bipolar cells RBCs). The corresponding maximum values of trial-averaged ΔF/Frest were significantly higher at ribbon-proximal locations than those at ribbon-distal locations, with mean ± SEM of 1.9±0.3 and 1.5±0.25, respectively (paired t-test, p<0.001, N=24). (A inset) The temporal profile of events between 20–60 ms is shown in an expanded view for better visualization. Scale bars: vertical, 0.5 (ΔF/Frest); horizontal,10 ms. (B) Spatially averaged Cal520LA fluorescence as a function of time at ribbon proximal (light cyan) and distal (dark cyan) locations. Data points show the average intensity (± SEM) in each horizontal row of 5 pixels for three 10 ms depolarizations at distinct ribbon locations (see Materials and methods and Figure 1). Fluorescence intensity is normalized with respect to the baseline fluorescence before stimulation, and averaged over all pixels (i.e. over space and time). The arrow indicates the onset of the 10 ms depolarizing stimulus. (n=21 ribbons from 4 different RBCs). The peak ΔF/Frest at the membrane after 10 ms depolarization was significantly larger for proximal than distal signals (paired t-test, ΔF/Frest: 3.1±0.4 and 1.9±0.2, respectively, p=0.001, N=21). The currents were not significantly different between the Cal520HA and Cal520LA conditions (unpaired t-test, Cal520HA average current = 45.1±4.5 pA; Cal520LA average current = 51.4±4.4 pA; p=0.33). (B inset) Temporal profile of events between 20–60 ms was expanded for better visualization. Scale bars: vertical, 0.5 (ΔF/Frest); horizontal, 10 ms.
Figure 3
Temporal properties of Ca2+ transients recorded with free and ribeye-bound Ca2+ indicators.
(A–B) Confocal images of the isolated RBCs that were whole-cell voltage-clamped using an internal solution containing the TAMRA-RBP (magenta) and either (A) Cal520HA-RBP (cyan) or (B) Cal520LA-RBP (cyan). Note prominent spots in both TAMRA-RBP and ribeye-bound Ca2+ indicators (A) Cal520HA-RBP (cyan) or (B) Cal520LA-RBP, showing the location of the ribbon. Scale bars, 2 μm. (C–D) Spatially averaged fluorescence of (C) Cal520HA-RBP (n=19) or (D) Cal520LA-RBP (n=30) as a function of time at ribbon proximal (light cyan), and distal (dark cyan) locations. Data points show the average intensity (± SEM) in each horizontal row of five pixels for 10 ms depolarizations at distinct ribbon locations. Fluorescence intensity at the onset of the 10 ms depolarizing stimulus (arrow) was normalized to the baseline fluorescence before stimulation and averaged over all pixels (i.e. over space and time). The amplitude differences between ribbon-proximal and ribbon-distal Ca2+ signals were well-resolvable using the ribbon-bound Cal520HA-RBP indicator (C, light vs. dark cyan traces, paired t-test: ΔF/Frest = 3.0±0.4 vs 1.9±0.3, respectively, p=0.001) and Cal520LA-RBP indicator (D, light vs. dark cyan traces, paired t-test: ΔF/Frest = 5.5±0.9 vs 3.3±0.8, respectively, p=0.003). The current amplitudes were not significantly different between Cal520HA-RBP and Cal520LA-RBP readings (mean current amplitudes: Cal520HA-RBP=49.8±2.2 pA, Cal520LA-RBP=49.9±2.9 pA; p=0.99). (C–D inset) Temporal profile of events between 10–50 ms were expanded for better visualization. Scale bars: vertical, 1 (ΔF/Frest, C inset) or 2 (ΔF/Frest, D inset); horizontal, 10 ms. (E–F) Average fluorescence intensity of (E) proximal and (F) distal Ca2+ signals obtained with Cal520LA-RBP (light cyan and dark cyan, respectively) and Cal520LA-free (gray). The amplitudes of ribbon-proximal Ca2+ signals were higher when measured with Cal520LA-RBP than with Cal520LA-free (E, Cal520LA-RBP (light cyan) vs. Cal520LA-free (gray), unpaired t-test: ΔF/Frest = 5.5±0.9, n=30 vs 3.1±0.4, n=21, p=0.04) but this was not the case for distal Ca2+ signals (F, Cal520LA-RBP (light cyan) vs. Cal520LA-free (gray), unpaired t-test: ΔF/Frest = 3.3±0.8, n=30 vs 1.9±0.2, n=21, p=0.15). (E–F inset) Events between 10–50 ms were expanded for better visualization. Scale bars: vertical, 2 (ΔF/Frest, E inset) and 1 (ΔF/Frest, F inset); horizontal, 10 ms.
Figure 4
Ca2+ signals at synaptic ribbon at different distances from the plasma membrane.
(A) Ultrastructure of a zebrafish rod bipolar cell (RBC) with kinetically distinct vesicle pools, as described in Figure 1—figure supplement 1 ultrafast releasable pool (UFRP) (green vesicles), and readily releasable pool (RRP) (yellow vesicles) are primarily released via ribbon sites (R) at the cytomatrix of the active zone (arrowhead). Recycling pool (RP, orange vesicles) in the cytoplasm (C), likely to be released via non-ribbon (NR) sites. Scale bar: 500 nm. (B) Representative x-t plots show the fluorescence intensity of Cal520LA-RBP (cyan) and TAMRA-RBP (magenta) as a function of distance (vertical axis) and time (horizontal axis) at (Ba) ribbon sites and (Bb) non-ribbon (NR) locations. The darker region at the upper edge of each plot is the extracellular space and the arrowheads show the timing of depolarization. Scale bars: vertical, 1.6 μm and horizontal, 75 ms. (C) Spatially averaged Cal520LA-RBP as a function of time at ribbon proximal (light cyan), distal (dark cyan), and non-ribbon locations (blue) (n=26 (proximal ribbons), 26 (distal ribbons), and 15 (non-ribbon) from 5~8 different RBCs, respectively). (C inset) Temporal profile of events between 0–100 ms were expanded for better visualization. Scale bars: vertical, 0.2 (ΔF/Frest); horizontal, 20 ms. (D) Ca2+ measurements along the ribbon axis using the nanophysiology approach demonstrate that the proximal Ca2+ signals can go as high as 26.4±3.1 µM (light cyan, N=26) and distal as 15.6±1.5 µM (dark cyan, N=26), and non-ribbon 10.4±0.4 µM (NR, blue, N=15), respectively, in response to 10 ms stimuli. Error bars show standard errors. All conditions were significantly different from each other as assessed by paired t-test when comparing proximal vs. Distal and unpaired t-test when comparing non-ribbon to proximal or distal (proximal vs. distal p=0.004; proximal vs. non-ribbon p<0.0001; distal vs. non-ribbon p=0.002). The currents were not significantly different between conditions (Mean current: 0.2 mM EGTA Cal520LA-RBP proximal and distal = 51.8±3.2 pA, 0.2 mM EGTA Cal520LA-RBP non-ribbon=47.3±3.0 pA; p=0.35).
Figure 5 with 1 supplement
Effect of exogenous Ca2+ buffers on spatiotemporal properties of Ca2+ microdomains in rod bipolar cell (RBC) terminal recorded with low-affinity ribbon-bound dye.
(A–D) Average temporal fluorescence intensity (normalized to ΔF/Frest) of proximal (light cyan) and distal (dark cyan) Ca2+ signals with Cal520LA-RBP as a function of time at distinct ribbon locations with pipette solution containing (A) 0.2 mM EGTA, (B) 2 mM EGTA, (C) 10 mM EGTA, or (D) 2 mM BAPTA. Proximal measurements were significantly higher than distal measurements in all conditions as assessed using paired t-tests (0.2 mM EGTA: p=0.0027, n=30; 2 mM EGTA: p=0.034, n=21; 10 mM EGTA: p<0.001, n=43; 2 mM BAPTA: p=0.0073, n=20). The currents between conditions were not significantly different from each other (mean current amplitudes in 0.2 mM EGTA: 50.6±3.0 pA, 2 mM EGTA: 49.7±3.1 pA, 10 mM EGTA: 47.3±3.1 pA, 2 mM BAPTA: 56.1±2.6 pA; 0.2 mM EGTA vs 2 mM EGTA: p=0.84, 0.2 mM EGTA vs 10 mM EGTA: p=0.46, 0.2 mM EGTA vs 2 mM BAPTA: p=0.19). Inset. The temporal profiles of events between 0–50 ms were expanded for better visualization. Scale bars: vertical, 2 (ΔF/Frest; panels A and B) and 1 (ΔF/Frest; panels C and D); horizontal, 10 ms. (E) Average temporal fluorescence intensity (normalized to ΔF/Frest) of proximal Ca2+ 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 ΔF/Frest was significantly higher with 2 mM Cal520LA-bound BAPTA than 2 mM Cal520LA-free BAPTA, with mean ± SEM of 3.6±0.9 and 1.4±0.2, respectively (unpaired t-test, p=0.028; Cal520LA-free: n=20; Cal520LA-bound: n=20). The currents between conditions were not significantly different from each other (mean current amplitudes in 2 mM BAPTA Cal520LA-RBP: 56.1±2.6 pA; 2 mM BAPTA Cal520LA-Free: 53.3±2.2 pA; p=0.42).
Figure 5—figure supplement 1
Spatiotemporal properties of Ca2+ microdomains along the synaptic ribbon in the rod bipolar cell (RBC) terminal.
(Aa–Da) Average temporal fluorescence intensity (normalized to ΔF/Frest) of proximal (light cyan), and distal (dark cyan) Ca2+ signals measured with Cal520HA-RBP as a function of time for distinct ribbon locations with pipette solution containing (A) 0.2 mM EGTA, (B) 2 mM EGTA, (C) 10 mM EGTA, or (D) 2 mM BAPTA. Statistics comparing proximal vs. distal in each condition can be found in Supplementary file 2 below. The currents were not significantly different between conditions. Mean current amplitude in 0.2 mM EGTA: 49.8±2.2 pA, 2 mM EGTA: 53.5±2.5 pA, 10 mM EGTA: 43.1±2.3 pA, 2 mM BAPTA: 45.6±4.2 pA; 0.2 mM EGTA vs. 2 mM EGTA: p=0.29, 0.2 mM EGTA vs. 10 mM EGTA: p=0.059, 0.2 mM EGTA vs. 2 mM BAPTA: p=0.39. (Ab–Db) The temporal profile of events between 10–50 ms (Ab and Db) and 5–45 ms (eb and hb) was expanded for better visualization. Scale bars: vertical, 1 (ΔF/Frest); horizontal, 10 ms.
Figure 6 with 1 supplement
Geometry of the computational model of intra-terminal Ca2+ dynamics.
Simulation domain is a box with dimensions (1.28×1.28 ×1.1) µm3, approximating the fraction of synaptic terminal volume per single ribbon. Ca2+ ions enter near the base of the ellipsoidal ribbon at four locations marked by black disks representing Ca2+ channels or their clusters. The semi-transparent gray coordinate plane Y=0 corresponds to the section used for the pseudo-color 2D [Ca2+] plots in Figure 7. Ca2+ is extruded on all surfaces of this domain, simulating combined clearance by pumps and exchangers on the plasmalemmal as well as internal endoplasmic reticulum and mitochondrial membranes.
Figure 6—figure supplement 1
Simulated effect of the endogenous mobile buffer of different concentrations on [Ca2+] dynamics in response to a 10 ms pulse.
(A) Estimation of Ca2+ current per ribbon. The average Ca2+ 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 Ca2+ current of 1 pA/ribbon. (B–C) Simulation results are analogous to Figure 7, except that no exogenous buffer is included, and the endogenous buffer is mobile, with a diffusion coefficient of 0.05 µm2/ms. Endogenous buffer concentrations were (A) 200 µM (resting buffering capacity 100) or (B) 1.44 mM (resting buffering capacity 720).
Figure 7
Simulation of the effect of an endogenous immobile buffer of different concentrations on [Ca2+] dynamics in response to a 10 ms pulse.
(A1–C2) In each sub-plot, the left-hand panels show the [Ca2+] time course in response to a 10 ms constant current pulse (total current of 1 pA) at five select locations marked by colored circles in the right panel. The right-hand panels show a pseudo-color plot of [Ca2+] in a 2D section of the 3D simulation volume in Figure 6, at a fixed point in time corresponding to the end of the current pulse. Concentration values for each level curve are indicated in the color bar. Endogenous buffer is immobile, with a concentration of 200 µM in panels A1-C1 (resting buffering capacity 100 µM), vs. 1.44 mM in panels A2-C2 (resting buffering capacity 720). Exogenous buffer concentrations were 0.2 mM EGTA (panels A1, A2), 10 mM EGTA (panels B1, B2), 2 mM BAPTA (panels C1, C2).
Figure 8 with 3 supplements
Heterogeneity in the spatiotemporal properties of Ca2+ microdomains in rod bipolar cell (RBC) terminal.
(A,Top panel) Cartoon of two representative RBCs (cell A and cell B), each containing differently colored ribbons. Bottom panel. Ribbon-to-ribbon variability was measured by recording local Ca2+ signals near different ribbons (yellow and purple traces) for each RBC. If an RBC had multiple readings for a single ribbon, averages were obtained for comparisons as described in Figure 8—figure supplement 2. Bottom panel inset: sample Ca2+ currents for the cells from which the Ca2+ signal sample traces mentioned above were obtained (purple and yellow traces). Currents were similar across the different cells. Vertical scale = 80 pA, horizontal scale = 5 ms. (B–C) Variability of Ca2+ signals in different ribbons across different cells in the presence of 10 mM EGTA, recorded using Cal520LA-RBP. Proximal Ca2+ amplitude values were significantly different between cells A and B (p=0.015), cells A and D (p=0.016), and cells B and D (p=0.004) but similar between A and C (n=4 cells, 4 fish), as assessed by Welch’s ANOVA with the Games-Howell post-hoc test. (C) In distal locations, ribbon amplitude values were significantly different between cells A and B (p=0.023) and cells B and C (p=0.049), but similar across all other cell comparisons (n=4 cells, 4 fish), as assessed by Welch’s ANOVA with the Games-Howell post-hoc test. The currents were not significantly different across the different cells, as assessed by unpaired t-tests (mean currents in RBC a: 47.7±2.9 pA, RBC b: 43.7±4.0 pA, RBC c: 43.4±2.0 pA, RBC d: 40.9±2.4 pA; RBC a vs. RBC b: p=0.45, RBC a vs. RBC c: p=0.22, RBC a vs. RBC d: p=0.14, RBC b vs. RBC c: p=0.93, RBC b vs. RBC d: p=0.54, RBC c vs. RBC d: p=0.52).(D, Top panel) Illustration of a RBC containing three ribbons (numbered 1–3). Bottom panel. Ca2+ signal measurements from three distinct ribbons (black, gray, and blue traces) were compared to determine the ribbon-to-ribbon variability within each RBC, as described in Figure 8—figure supplement 3. Bottom panel inset: sample Ca2+ currents for the cells from which the Ca2+ signal sample traces mentioned above were obtained (black, gray, and blue traces). Currents were similar across the different cells. Vertical scale = 80 pA, horizontal scale = 5 ms. (E–F) Box plot illustrating [Ca2+] across various ribbons of an individual RBC, which is shown as RBC a. Ribbon variability within individual cells was measured with 10 mM EGTA using Cal520LA-RBP at (E) proximal and (F) distal locations. (E) Proximal Ca2+ amplitude values were significantly different among all ribbons (paired t-test, p<0.001) (n=5 ribbons, 1 RBC, 1 fish). (F) Distal Ca2+ amplitudes were significantly different among all ribbon comparisons (paired t-test, p<0.001) except for ribbons 2 and 5 (n=5 ribbons, 1 RBC, 1 fish). Similar analyses were conducted in two more cells and found similar observations (data not shown). The currents were similar across all ribbons since these were readings from the same cell. Given that some ribbons only have one reading, it is not possible to conduct a paired t-test to statistically compare them; however, the average current ± standard error for the cell shown was 47.7±2.9 pA.
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Figure 8—source data 1
Data presentation for ribbon variability between cells.
Table explaining how the data is presented in Figure 8A–C.
- https://cdn.elifesciences.org/articles/105875/elife-105875-fig8-data1-v1.docx
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Figure 8—source data 2
Data presentation for ribbon variability within individual cells.
Table explaining how the data is presented in Figure 8D–F.
- https://cdn.elifesciences.org/articles/105875/elife-105875-fig8-data2-v1.docx
Figure 8—figure supplement 1
Variability in Ca2+ transients in response to brief stimuli.
(A–B) Spatially averaged Cal520HA fluorescence as a function of time at ribbon proximal location. The average (black trace) of three stimuli (gray traces) at a single ribbon active zone/Ca2+ microdomain was obtained from two rod bipolar cells (RBCs) as described in Figure 2A. Note the amplitude variability between two cells (#1, panel a and #2, panel b) with similar Ca2+ currents.
Figure 8—figure supplement 2
Illustration to demonstrate ribbon variability between cells.
An example of two rod bipolar cells, each containing three ribbons (the first cell has ribbons depicted in pink, purple, and orange; the second cell has ribbons depicted in blue, green, and brown).
Figure 8—figure supplement 3
Illustration to demonstrate ribbon variability within individual cells.
Example of a rod bipolar cell containing three ribbons (depicted in pink, purple, and orange).
Figure 9
Distribution of measured distance between synaptic ribbons in three zebrafish RBCs.
(A) Reconstruction of three RBC terminals closest to the ganglion cell layer resembling the shape and size of the mammalian RBC1. The ribbons are shown in magenta. Note that the total number of ribbons includes the ‘floating’ ribbons detached from the plasma membrane. RBCI and RBC II contained 1 and 8 floating ribbons, respectively. Scale bar, 5 µm. RBCs, rod bipolar cells. (B) Overview of the distance measurements. Top. 3D rendering of a single RBC terminal is shown in light green with ribbons in magenta. Black lines show an example of the different serial block face scanning electron microscopy (SBF-SEM) layers that are cut to obtain individual images. Bottom. Four sample SBF-SEM layers are shown with two example ribbons (magenta) located near each other but in different layers. If two ribbons were on the same layer, their linear distance was taken, whereas if they were located in different layers, their linear distance and height difference were used to calculate their actual distance using the Pythagorean Theorem. Each SBF-SEM layer has a thickness of 50 nm. A 3D volume movie of the synaptic terminal of a zebrafish bipolar neuron synaptic ribbon distribution and measurement is provided in Video 1.(C) Histograms showing the ribbon distance for the three RBCs. x0 shows the mean of the distribution. (D) Histogram of the distances between each ribbon and its five nearest ribbons for all ribbons contained in the three RBCs. x0 shows the mean of the distribution. Please note that the y-axis for B and C have different sizes, given that they represent the number of occurrences of each event.
Figure 10 with 1 supplement
Serial block-face scanning electron microscopy analysis reveals heterogenous rod bipolar cell (RBC) ribbon shape, size, and area of the ribbon facing the plasma membrane.
(A–C) Electron microscopy (EM) images of zebrafish RBC ribbon structures (A) and their respective 3D reconstructions to illustrate different shapes and sizes of synaptic ribbons (A, B, and C, magenta), plasma membrane (A and B, yellow), and the area of the ribbon facing the plasma membrane (A, B, and C, cyan). Each of the five rows of images illustrates one ribbon synapse from a zebrafish retinal RBC. A 3D reconstruction of the RBC synaptic terminal and ribbon from serial block face scanning electron microscopy (SBF-SEM) stacks is provided in Video 2. (D) 3D reconstruction of the RBC terminal closest to the ganglion cell layer resembles the shape and the size of the mammalian RBC1 (Hellevik et al., 2024). The ribbons are colored magenta. (E) Summary of the active zone size, the area of the ribbon associated with the plasma membrane measured in serial sections across the three RBCs from Figure 9A. The individual distribution of the three RBCs active zone sizes is provided in Figure 10—figure supplement 1. The z-size of the SBF-SEM sections is 50 nm, and each ribbon spans 2–5 consecutive sections. The solid cyan circles in the violin plots show individual synaptic ribbon measurements from three RBCs, with the average measurements shown in the solid black circle. Note that floating ribbons are not shown since they were not attached to the plasma membrane.
Figure 10—figure supplement 1
Three rod bipolar cells (RBCs) active zone reconstructed from serial block-face scanning electron microscopy.
The box and whisker plots summarize the area of the individual ribbon associated with the plasma membrane measured in serial sections of each of the three RBCs from main Figure 9A. The solid cyan circles in the box and whisker plots show the measurements of individual synaptic ribbon measurements, with the average shown as a solid black square and median values as horizontal black dotted lines. The boxes represent the 25th-75th percentiles tests. Note that RBCI and RBCII contained 1 and 8 floating ribbons that were not included in the main Figure 9A and since they were not attached to the plasma membrane.
Figure 11
Heterogeneity of Ca2+ microdomains in rod bipolar cell (RBC) terminals.
Larger ribbons have stronger maximal Ca2+ influx and larger CAZ. (A) Images show voltage-clamped RBC filled with a solution containing TAMRA-RBP to label ribbons before depolarization (left, magenta) and Cal520LA-RBP to measure the amplitude of Ca2+ influx (middle, cyan) during depolarization and superimposed of the two (right) to compare the size of ribbon vs. maximal Ca2+ influx. A time-lapse movie of the synaptic terminal of a zebrafish bipolar neuron during Ca2+ influx is provided in Video 3. (B) Scatter plot of maximal Ca2+ influx (F/Frest) vs. TAMRA-RBP. Dashed lines are linear regressions, and r is Pearson’s correlation coefficient. (C) Scatter plot of maximal synaptic ribbon length vs. maximal synaptic ribbon width as estimated from SBF-SEM images. Dashed lines are linear regressions, and r is Pearson’s correlation coefficient. (D) Scatter plot of area of AZ vs. ribbon width (filled black circles) and of area of AZ vs. ribbon length (filled gray circles). All the ribbons are included in the analysis. Dashed lines are linear regressions, and r is Pearson’s correlation coefficient. AZ, Active Zone.
Videos
Video 1
3D volume movie of the synaptic terminal of a zebrafish rod bipolar cell terminal showing the distribution of included synaptic ribbons and measurements.
Rod bipolar cell (RBC) terminal is shown in light green with ribbons in magenta. Illustrations show how the measurements were obtained for ribbons on the same vs. different layers.
Video 2
A 3D reconstruction of the rod bipolar cell (RBC) synaptic terminal and ribbon from serial block face scanning electron microscopy (SBF-SEM) stacks.
(A) 3D reconstruction of the RBC terminal (green) with included synaptic ribbons (colored magenta). (B) 3D reconstruction of the RBC synaptic ribbons shows different shapes and sizes of ribbon (magenta) and the AZ, the area of the ribbon facing the plasma membrane (cyan). AZ, active zone.
Video 3
A time-lapse movie of the synaptic terminal of a zebrafish bipolar neuron during Ca2+ influx.
Left: Rod bipolar cell (RBC) terminal labeled with TAMRA-RBP shows the locations of synaptic ribbons (magenta spots). Middle: RBC terminal filled with Cal520LA-RBP shows the Ca2+ influx in 0.02 s, looped two times to see Ca2+ influx in 0.07 s. Right: Superimposed TAMRA-RBP and Cal520LA-RBP shows the Ca2+ influx as spot-like maxima near the membrane during depolarization. TAMRA-RBP 10 images before depolarization, and stacks of Cal520LA-RBP five images before depolarization and five images during depolarization were compiled to demonstrate the locations and magnitude of the Ca2+ influx. The interval between frames is 407 ms, total duration of the video 0.9 s. Each frame is an individual, unaveraged image.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Danio rerio) | WIK | ZIRC | ZDB-GENO-010531–2 | |
| Other | Hyaluronidase, type V | Sigma | H6254 | Enzyme (1100 units/mL) |
| Chemical compound, drug | NaCl | Fisher Scientific | S271-3 | 120 mM |
| Chemical compound, drug | KCl | Fisher Scientific | P217 | 2.5 mM |
| Chemical compound, drug | CaCl2 | Honeywell / Fluka | 21117 | 0.5 mM |
| Chemical compound, drug | MgCl2 | Fisher Scientific | AM95305 | 1 mM/3 mM |
| Chemical compound, drug | Glucose | Sigma | G5146 | 10 mM |
| Other | HEPES | J.T. Baker | 4018–04 | Buffer |
| Chemical compound, drug | DL-cysteine | Fluka | 30197 | 2 mM |
| Other | Papain | Fluka | 76220 | Enzyme (20–30 units/mL) |
| Other | Fire-polished glass Pasteur pipette | Fisher Scientific | 03-678-20A | fire-polished glass triturates |
| Other | RBP- Tetramethylrhodamine (TAMRA) | LifeTein LLC, NJ | GIDEEKPVDLTAGRRAG | Peptide |
| Other | Cal520HA-free High-affinity | AAT Bioquest | 21140 | Calcium indicator, Kd 320 nM |
| Other | Cal520LA-free Low-affinity | AAT Bioquest | 20642 | Calcium indicator, Kd ~90 µM |
| Other | Cal520HA-RBP | LifeTein | Ca2+ indicator: 20610 RBP: NH2-CIEDEEKPVDLTAGRRAC-COOH | Peptide Kd 320 nM |
| Other | Cal520LA-RBP | AAT Bioquest | Ca2+ indicator: 20611 RBP: NH2-CIEDEEKPVDLTAGRRAC-COOH | Peptide Kd ~90 µM |
| Chemical compound, drug | Cs-gluconate | Hello Bio, Incorporation | HB4822-10g | 120 mM |
| Chemical compound, drug | tetraethyl-ammonium-Cl | Tocris | 3068 | 10 mM |
| Chemical compound, drug | N-methyl-d-glucamine-EGTA | NMDG, Sigma EGTA, EMD Millipore | M2004 324626 | 0.2 mM |
| Chemical compound, drug | Na2ATP | Fisher Scientific | BP413 25 | 2 mM |
| Chemical compound, drug | Na2GTP | Fisher Scientific | 10106399001 | 0.5 mM |
| Chemical compound, drug | Ames’ medium | US Biological Life Sciences | A1372-25 | buffer |
| Other | Glutaraldehyde 4% in 0.1 M Sodium-cacodylate buffer | Electron Microscopy | 16539–06 | buffer |
| Other | Olympus laser-scanning confocal microscope | Olympus, Shinjuku, Tokyo, Japan | Model IX 83 motorized inverted FV3000RS | microscope |
| Other | Electron microscopy system | Carl Zeiss Microscopy GmbH Jena, Germany | Zeiss 3-View | microscope |
| Software, algorithm | PatchMaster | HEKA Instruments, Inc, Holliston, MA | Version v2x90.4 | |
| Software, algorithm | Fluoview FV31S-SW Software | Olympus, Center Valley, PA | Version 2.3.1.163 | |
| Software, algorithm | Fiji/ImageJ, | https://imagej.nih.gov/ | Version 2.16.0/1.54 P | |
| Software, algorithm | Igor Pro Software | Wavemetrics, Portland, OR | Version 9.05 | |
| Software, algorithm | Microsoft Excel | Microsoft | Version 16.81 | |
| Software, algorithm | R Studio | R Studio | Version 2023.09.0+463 | |
| Software, algorithm | Adobe Photoshop | Adobe | Version 25.9 | |
| Software, algorithm | TrakEM plugin of ImageJ | https://imagej.nih.gov/ | Version 1.5 h |
Additional files
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Supplementary file 1
Effect of exogenous Ca2+ chelators alter Ca2+ signals gradient along synaptic ribbon measured with Cal520LA-RBP.
There were significant differences between proximal vs distal measured as ΔF/Frest, in all conditions, as found through paired-sample t-test analysis performed on RStudio. Differences were smaller between proximal vs distal Ca2+ signals in 0.2 mM, 2 mM, and 10 mM EGTA conditions, but more prominent with 2 mM BAPTA (0.2 mM EGTA: proximal vs distal: p=0.0027, 2 mM EGTA: proximal vs distal: p=0.034, 10 mM EGTA: proximal vs distal p=0.00013, 2 mM BAPTA: proximal vs. distal: p=0.0073, n=22).
- https://cdn.elifesciences.org/articles/105875/elife-105875-supp1-v1.docx
-
Supplementary file 2
Effect of exogenous Ca2+ chelators alters the Ca2+ signal gradient along the synaptic ribbon measured with Cal520H-RBP.
There were significant differences between proximal vs distal measured as ΔF/Frest, in all conditions as found through paired-sample t-test analysis performed on RStudio. Differences were smaller between proximal vs distal Ca2+ signals in 0.2 mM EGTA and 2 mM EGTA conditions, but more prominent with 10 mM EGTA, and further enhanced with 2 mM BAPTA (0.2 mM EGTA: proximal vs distal p=0.00135, n=19; 2 mM EGTA: proximal vs distal p=7.4∙10–4, n=23; 10 mM EGTA: proximal vs distal p=1.4∙10–5, n=29; 2 mM BAPTA: proximal vs distal p=0.0046, n=22).
- https://cdn.elifesciences.org/articles/105875/elife-105875-supp2-v1.docx
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Supplementary file 3
Model parameters for Ca2+ diffusion, buffering, and clearance.
Simulations were performed assuming an endogenous buffer with a total concentration of either 1.4 mM total resting buffering capacity of 720 Oesch and Diamond, 2011; Burrone et al., 2002; Coggins and Zenisek, 2009, or a lower concentration of 200 µM corresponding to a buffering capacity of 100. Simulations in Figure 7 assumes immobile endogenous buffer, while this file assumes a typical value of buffer mobility of 0.05 µm2/ms. The Ca2+ clearance parameters are adapted from Graydon et al., 2011; Jarsky et al., 2010; Mennerick and Matthews, 1996; Singer and Diamond, 2003; Snellman et al., 2009; Von Gersdorff and Mathews, 1994; Augustine et al., 1991. Note that flux units of (µM µm)/ms are equivalent to 10–21 mol/(µm2ms)=602 ions/(µm2ms). Properties of EGTA and BAPTA (not listed here) are summarized in Burrone et al., 2002.
- https://cdn.elifesciences.org/articles/105875/elife-105875-supp3-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/105875/elife-105875-mdarchecklist1-v1.pdf
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Nanophysiology approach reveals diversity in calcium microdomains across
eLife 14:RP105875.
https://doi.org/10.7554/eLife.105875.4
