Synaptic vesicles labeled with Vglut1-paRFP stably associate with synaptic ribbons at the active zone.(A) Stereogram of a 3D reconstruction from a series of confocal optical sections through the synaptic terminal of a zebrafish BPC, showing photoactivated Vglut1-paRFP (red) near synaptic ribbons (green). A rotating view of the reconstruction is provided in Video 2. Scale bar = 2 µm. Details of recording and photoactivation are provided in Figure 1-figure supplement 1. (B) Close-up view of a synaptic ribbon labeled with green-fluorescent RBP, which also fills the cytoplasm. Outside of the cell is to the right, which is the standard orientation adopted for all images. Average of 16 frames. (C) View of the same region showing a single Vglut1-paRFP spot, averaged over 128 frames. (D) Superposition of images from B and C. (E) 2D Gaussians (Figure 1-figure supplement 2) fitted to RBP and paRFP fluorescence, with peaks marked by + and X respectively. (F) Positions of 81 paRFP spots (red Xs) relative to the center of the RBP-labeled ribbon, superimposed on an electron micrograph of a ribbon in a zebrafish BPC. The pink region depicts the ~100-nm region imaged in TIRFM experiments (except that the region would be planar in an actual TIRFM experiment). The inset reproduces the ribbon image without overlaid paRFP centroids. (G) Ribbon image with superimposed positions of a paRFP spot, shown by Xs, followed for 90 s without stimulation. Each position was determined by fitting a 2D Gaussian to a single averaged image of paRFP fluorescence like that shown in H. (H) An example of a single paRFP image, averaged over 10 frames, used to estimate paRFP positions shown in G. (I) Stability of paRFP location in the absence of stimulation for 10 experiments like that illustrated in G and H. Each symbol shows an individual paRFP location, and the different symbol types show results for a particular experiment.

Overview of the experimental procedure.(A) Retinas were removed from transgenic zebrafish that expressed Vglut1-paRFP fusion protein after heat shock. (B) Intact, acutely dissociated retinal BPCs were voltage-clamped via a whole-cell patch pipette placed on the synaptic terminal. (C) Electron micrograph showing the synaptic terminal of a zebrafish BPC, with the boxed region around a ribbon at higher magnification in the inset. Ribbons are surrounded by tethered synaptic vesicles. (D) Cartoon illustrating photoactivation of paRFP by 405-nm light. (E) Example of red fluorescence in the synaptic terminal before and after photoactivation.

Generation and analysis of line scan data. (A) Scan lines were positioned perpendicular to the plasma membrane, extending from the intracellular side of the ribbon into the extracellular space. For illustrative purposes, a second scan line is shown at a non-ribbon location. Lower-case letters show line positions for panels B-D. (B) Intensity profile of green RBP fluorescence at the non-ribbon location. The parameter x_1/2 from the sigmoid fit (black line) is taken as the position of the membrane (see Materials and methods). (C) Intensity profile of green RBP fluorescence at the ribbon. The parameter x₀ is the peak of the Gaussian fit, giving the x-position of the center of the ribbon (see Materials and methods). (D) Intensity profile of paRFP fluorescence at the ribbon, with the x-position of the labeled vesicle given by x₀. (E) Example of x-t image of paRFP spot at a ribbon, consisting of 64 line scans (right) taken over 472 ms. X-axis intensity profiles are plotted (left) for the first 16 line scans to illustrate the noise within and across individual line scans. The superimposed black line shows the average of all 64 lines. (F) Data from E were averaged over four lines to reduce noise and allow more precise localization of the paRFP spot along the x-axis. The intensity profiles show the first four of the temporally averaged line scans, with the average for all lines superimposed in black. (G) Example x-t images averaged over five line scans, showing a stable paRFP-labeled synaptic vesicle associated with a ribbon, which was labeled in green with RBP. Membrane potential was voltage-clamped at -60 mV. (H) Intensity profiles of RBP (green) and paRFP (red) taken in successive 406-ms intervals from the images in G. The red Xs show the position of x₀ for each paRFP trace, which varied over a range of 48 nm during the 15-s recording.

Movement and loss of vesicles during sustained depolarization.(A) Example x-t images in which a previously stable paRFP-labeled vesicle disappeared from view during sustained depolarization. The timing of depolarization and the evoked Ca²⁺ current are shown to the right. Analysis of x-t line scan images is described in Figure 3. (B) Example showing a paRFP-labeled vesicle that appeared distal to the center of the ribbon during sustained depolarization, moved toward the membrane, and disappeared along a Gaussian time course (Figure 4-figure supplement 1). (C) Fluorescence intensity profiles along the x-axis for the example in panel B, showing ribbon position (green) and paRFP positions at appearance and disappearance (red). Black lines are fits described in Figure 3. The dotted line shows the estimated position of the plasma membrane estimated from x_1/2 obtained from the fit to RBP fluorescence. (D) Displacement amplitude for 88 paRFP-labeled vesicles along the x-axis prior to disappearance, as a function of initial starting position relative to the center of the ribbon. Positive and negative displacements are movements toward and away from the membrane, respectively. Open circles show the average of groups of five points binned by starting position. Error bars: ± 1 sem. Positive starting positions are nearer the membrane, and negative positions are farther away. (E) Histogram of final positions of paRFP-labeled vesicles along the x-axis just before disappearance.

Synaptic vesicle exocytosis reported by pHluorin occurs at a distance from the plasma membrane. (A) Example of x-t images showing a pHluorin event from SypHy (green) at a ribbon shortly after onset of depolarization from -60 mV to -15 mV, shown by the trace to the right. Ribbon fluorescence from deep-red CF633-RBP is pseudocolored red. (B) Example of x-t images showing a pHluorin event from SypHy (green) on the membrane-distal side of a ribbon (red) in a different cell during depolarization to -15 mV. (C) (D) Fluorescence intensity profiles along the x-axis for RBP (red) and pHluorin (green) for the examples shown in A and B. Black lines are fits as described in Figure 3A-D and in Materials and methods. The x-axis position of the pHluorin event was taken to be the peak of the Gaussian from the fit (x₀, shown by the dotted green line), and the position of the plasma membrane was taken to be the parameter x_1/2 (dotted black line) from the fit to the fluorescence profile of the ribbon (red traces). (E) Histogram of the x-axis position of 127 pHluorin events from SypHy and Vglut1-pHluorin during sustained depolarization. The solid arrow shows the average relative position of the plasma membrane, estimated as described in Materials and methods (error bar: ± 1 sem). The dashed arrow shows the estimated position of the membrane at the top and bottom of the optical section, arrived at by assuming that membrane curvature observed along the y-axis for each x-y image also applied in the z-axis. Most of the pHluorin events fell outside the range of membrane positions between the two arrows.