Neurons communicate with one another through junctions known as synapses. When a neuron is activated, it triggers the release of chemicals called neurotransmitters at the synapse, which bind to and activate neighbouring neurons. Neurons involved in vision, sound and balance contain “ribbon” synapses, which are able to release neurotransmitters steadily over longer periods of time than other types of synapse.

Neurotransmitters inside neurons are packaged into small structures called vesicles, which can then fuse with the cell’s surface membrane to release the neurotransmitters from the cell. Unlike other types of synapse, ribbon synapses are able to release these vesicles in a continuous fashion. How vesicles move around at the synapses remains poorly understood because monitoring the vesicles in living cells is technically difficult and previous studies were limited to tracking vesicles in a small part of the synapse. Now, Vaithianathan et al. overcome these technical hurdles to follow the movement of vesicles across whole ribbon synapses in zebrafish eyes.

The experiments use fluorescent proteins to track the movement of the vesicles under a microscope. Vaithianathan et al. find that vesicles at ribbon synapses move very little when the neurons are not active. However, when the neurons are activated, the vesicles that are near the cell membrane fuse with it and release their neurotransmitters. Other vesicles that are further away from the membrane then move to fill in the spaces vacated by the fusing vesicles.

Further experiments show that some of the vesicles that are further away from the membrane can fuse with vesicles that have already released their neurotransmitter but remain in place at the membrane. This process – known as compound fusion – allows neurotransmitters to be released over a longer period of time by providing a path for vesicles to release neurotransmitters without having to approach the membrane first. The next challenge is to develop a computational model using the data from this study to better understand how ribbon synapses work.

Neurons communicate at synapses, where chemical neurotransmitter is released from the presynaptic terminal by fusion of transmitter-containing synaptic vesicles with the plasma membrane. A macromolecular complex, the cytomatrix at the active zone (CAZ; Südhof, 2012) is thought to capture and organize synaptic vesicles to support neurotransmitter release by presynaptic terminals (Sigrist and Schmitz, 2011; Gundelfinger and Fijtova, 2012; Hallermann and Silver, 2013), but the dynamics of this process at living synapses remain largely elusive. In order to follow the trafficking of vesicles in real time at the CAZ, methods are needed to label and image both vesicles and the active zone at the same time in living synapses. To label the living active zone, we used fluorescent Ribeye-binding peptide (RBP; Zenisek et al., 2004), which marks the synaptic ribbon found at the active zones of retinal bipolar cells (BPCs) and other neurons that release neurotransmitter continuously in response to graded changes in membrane potential (Matthews and Fuchs, 2010). However, monitoring the trafficking of tiny synaptic vesicles (30–50 nm in diameter) is a technical challenge, which we were able to meet by targeting a photoactivatable fluorescent protein to synaptic vesicles and using super-resolution photoactivated localization microscopy (Betzig et al., 2006) to track movements of single vesicles in voltage-clamped synaptic terminals. We also targeted the exocytosis reporter pHluorin (Sankaranarayanan et al., 2000) to synaptic vesicles (Granseth et al., 2006; Voglmaier et al., 2006) in order to detect vesicle fusion. These approaches then allowed us to analyze the trafficking and fusion of vesicles at the active zone of BPC ribbon synapses during ongoing neurotransmitter release.

Previously, studies of synaptic vesicle trafficking and fusion at ribbon synapses of BPCs have been carried out using total internal reflection fluorescence microscopy (TIRFM) to image single vesicles labeled with FM dye (Zenisek et al., 2000, 2002; Holt et al., 2004; Midorikawa et al., 2007; Zenisek, 2008; Joselevitch and Zenisek, 2009). Some of the conclusions from this work are: 1) vesicles stably associate with ribbons in the absence of stimulation (‘residents’), 2) these resident vesicles rapidly undergo exocytosis in response to depolarization, and 3) new vesicles (‘newcomers’) appear, move toward the membrane, and fuse during sustained depolarization. Although TIRFM is a powerful approach to study membrane-associated phenomena, it is limited to imaging labeled vesicles within ~100 nm of the plasma membrane (e.g., Zenisek et al., 2000), which is insufficient to provide coverage of all the ribbon-associated vesicles. The method also requires tight adherence of the plasma membrane to a planar substrate, which restricts observations to a small part of the terminal and eliminates the natural membrane curvature in that observable region. Furthermore, to our knowledge, only Midorikawa et al. (2007) and Zenisek (2008) have combined vesicle imaging with ribbon labeling, and then only in sequentially acquired images separated by some time, which were intended to test whether observed hotspots of fusion coincided with ribbons. Because of these limitations, we used two-color laser scanning methods that allowed single labeled vesicles to be observed 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. Since the positions of the ribbon and a labeled vesicle were known accurately, we were able to detect vesicle movements on the ribbon prior to fusion and determine where vesicles resided on the ribbon when they fused. Therefore, our experiments complement and significantly extend the previous studies based on TIRFM.

To track single vesicles, we targeted the photoactivatable fluorescent protein paRFP (Subach et al., 2010) to synaptic vesicles by fusing it to the C-terminus of the vesicle membrane protein Vglut1 (vesicular glutamate transporter 1). The resulting fusion protein was then inducibly expressed in transgenic zebrafish (Figure 1; Figure 1—figure supplement 1). Synaptic vesicles bearing Vglut1-paRFP participated normally in the vesicle cycle, since they were labeled by activity-dependent uptake of FM dye. By photoactivating Vglut1-paRFP at low density, we ensured that a field of view contained only a single fluorescent synaptic vesicle, which we then localized using high-resolution confocal microscopy (Figure 1A–F; Figure 1—figure supplement 2). Both mobile and immobile vesicles were visible after photoactivation (Video 1), but the immobile vesicles were closely associated with ribbons (Figure 1A,F; Video 2), which were labeled with green-fluorescent RBP introduced via a whole-cell patch pipette. Therefore, we conclude that the ribbon-associated vesicles are immobile because they are tethered to or near the ribbon itself (Figure 1F). These immobile vesicles likely correspond to the ‘resident’ vesicles observed at BPC active zones using TIRFM (Zenisek et al., 2000; Midorikawa et al., 2007), except that our laser-scanning method detected vesicles located everywhere on the ribbon instead of only in a region <100 nm from the plasma membrane (the region marked with pink shading for illustrative purposes in Figure 1F).

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Resident vesicles at ribbons move very little in the absence of stimulation (Zenisek, 2008), and we therefore used the temporal jitter in the observed position of paRFP-labeled vesicles relative to ribbons to estimate the spatial precision of the localization method under our imaging conditions. A series of x-y images was acquired while the synaptic terminal was voltage-clamped at -60 mV to prevent release, and after averaging over 4–10 frames to reduce noise, the positions of the paRFP spot and ribbon were determined by fitting 2D-Gaussians, as illustrated in Figure 1E. Vesicle position was then expressed relative to the center of the ribbon (i.e., the peak of the ribbon 2D-Gaussian) in order to control for small movements of the cell and/or ribbon during an experiment. The vesicle position estimated in this manner varied little during the 90-s imaging period (Figure 1G–I), within ranges that averaged 27 nm on the x-axis and 17 nm on the y-axis. Assuming that vesicles actually did not move during the imaging, we attribute the observed variation in estimated position to imaging noise that limits the spatial resolution. Therefore, genuine stimulus-induced vesicle movements on the ribbon of approximately a vesicle diameter would be resolvable by comparing averaged x-y images before and after stimulation.

Vesicle turnover and replenishment on ribbons

What happens to the immobile ribbon-associated vesicles during transmitter release? To control release, isolated BPCs were voltage-clamped via a whole-cell pipette placed directly on the synaptic terminal (Figure 1—figure supplement 1B) and stimulated by brief depolarization from -60 to 0 mV to activate voltage-gated calcium channels (Figure 2A, inset), causing calcium influx that triggered synaptic vesicle fusion. BPCs exhibit two kinetically distinct components of transmitter release during depolarization (for zebrafish BPCs, see Vaithianathan and Matthews, 2014): a fast component depleted within 10 ms, and a slower component lasting hundreds of ms. We first determined how individual paRFP-labeled vesicles at the ribbon responded when the system was perturbed by releasing just the fast component, which is thought to represent the cohort of vesicles tethered to the ribbon nearest the plasma membrane (Matthews and Fuchs, 2010), where voltage-gated calcium channels reside.

Figure 2

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On some trials after a 10-ms stimulus, a new stable paRFP spot appeared at the ribbon (Figure 2A,C), while on other trials, a previously present paRFP spot disappeared after the stimulus (Figure 2B,D), representing single-vesicle replenishment and loss, respectively. When the loci of gained and lost paRFP spots were determined as illustrated in Figure 1 and plotted with respect to the center of the ribbon (Figure 2E), there was no difference in the distribution of appearances and disappearances along the axis parallel to the membrane (the y-axis; p = 0.4, Wilcoxon rank test). However, disappearances clustered significantly closer to the plasma membrane than appearances (Figure 2E) along the axis perpendicular to the membrane (the x-axis; p<10^-8, Wilcoxon rank test). This pattern revealed by single-vesicle imaging is consistent with selective fusion of membrane-proximal synaptic vesicles during brief depolarization, but preferential recruitment of new vesicles at the opposite pole of the ribbon to replace those lost, as suggested previously from population behavior of vesicles labeled with FM dye (Vaithianathan and Matthews, 2014). A simple interpretation is that loss of membrane-proximal vesicles creates the opportunity for vesicles remaining on the ribbon to rearrange and occupy the vacated region, opening up 'slots' for new vesicles at more distal positions on the ribbon.

Vesicle dynamics during the sustained component of release

In addition to the rapid burst of release at the onset of depolarization, ribbon synapses also release neurotransmitter continuously during sustained depolarization. To examine vesicle trafficking during this sustained component of release evoked by depolarization from -60 mV to -10 mV for >0.5 s, we measured fluorescence along a short scan-line perpendicular to the plasma membrane, which yielded x-t raster plots that were analyzed as shown in Figure 3 to determine the x-axis position of paRFP spots relative to the synaptic ribbon and the plasma membrane (also see Image analysis section of Materials and methods). Although paRFP fluorescence was detectable in individual scan lines (Figure 3E), the noise level precluded accurate localization of the emitter by fitting the x-axis intensity profile of single lines. In the example shown in Figure 3E, for instance, the peak of the fitted Gaussian varied over a range of 170 nm in 16 consecutive individual lines. However, averaging across 4 scan lines reduced the range of variation of fitted positions to 20 nm, as shown in Figure 3F. As a result, we routinely averaged over 4–10 lines, depending on the noise level in a particular experiment, in order to achieve localization along the x-axis with a resolution of approximately one vesicle diameter. After such averaging, the position of a labeled vesicle in the absence of stimulation was stable for many seconds within a range of ± 25 nm (Figure 3G,H), which we take as the spatial precision of the line-scan method.

Figure 3

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We next examined changes in paRFP-labeled vesicles at ribbons during activation of calcium current elicited by depolarizing voltage-clamp steps from -60 to -15 mV for 500–5000 ms. During sustained depolarization, previously stable paRFP spots often disappeared from the ribbon, either without a change in position before loss (e.g., Figure 4A) or after moving toward the membrane (e.g., Figure 4B,C). Whether vesicles moved or not before disappearance depended on starting position with respect to the membrane (Figure 4D): vesicles nearer the plasma membrane (positive starting positions in Figure 4D) did not consistently move before disappearing, while distal vesicles (negative starting positions) moved toward the membrane before loss. Since membrane-proximal vesicles did not move while distal vesicles moved toward the ribbon center, the histogram of final paRFP positions just prior to disappearance was broad (Figure 4E) and covered the full range of ribbon-associated vesicle positions (cf., Figure 1F). During disappearance, paRFP fluorescence did not cease abruptly, as would occur for bleaching, but instead declined along a Gaussian time course, which is consistent with diffusion of the emitter away from the scanned region (Figure 4—figure supplement 1). It is uncertain at present whether this diffusion reflects movement of the Vglut1-paRFP protein itself, as would occur for example if the protein incorporated into the plasma membrane after vesicle fusion, or dissociation of an intact vesicle from the ribbon.

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Locus of vesicle fusion revealed by pHluorin reporter

Are vesicles that disappear from distal positions on the ribbon during sustained depolarization simply shed from the ribbon without participating in release, or do they somehow fuse and release their contents without approaching the plasma membrane? To detect the fusion of ribbon-associated vesicles, we generated transgenic zebrafish that express the exocytosis reporter SypHy (Granseth et al., 2006) (Synaptophysin-pHluorin fusion protein) or Vglut1-pHluorin (Voglmaier et al., 2006) under control of heat-shock promoter. Since pHluorin-fusion proteins mixed with pre-existing native vesicle proteins, pHluorin events were sparse during depolarization, allowing detection and localization of single events with respect to the ribbon, in the same manner as paRFP-labeled vesicles. For 2-color imaging, ribbons were labeled with deep-red CF633-RBP, which did not interfere with pHluorin fluorescence. Under these conditions, unitary pHluorin events representing vesicle fusion were observed in x-t raster plots during sustained depolarizing voltage-clamp steps from -60 to -15 mV for 500–5000 ms (Figure 5A,B), but not in the absence of stimulation. However, the signal-noise ratio for pHluorin fluorescence was poorer than for paRFP, and this necessitated averaging over a greater number of scan lines to achieve spatial resolution in the range of a vesicle diameter for pHluorin events, as illustrated in Figure 5—figure supplement 1. The fluorescence intensity profile of pHluorin events perpendicular to the membrane was then analyzed as described earlier for paRFP to determine the position of pHluorin events relative to the ribbon and the plasma membrane (Figure 5C,D). Events sometimes arose near the plasma membrane, suggesting fusion of membrane-proximal vesicles (e.g., Figure 5C), but events also commonly originated at the distal pole of the ribbon, at a distance from the plasma membrane (e.g., Figure 5D). The histogram of event positions relative to the center of the ribbon (Figure 5E) showed that pHluorin signals were broadly spread across the ribbon (Figure 5E) and were not confined to positions near the plasma membrane, as would be expected if vesicles must first approach the plasma membrane in order to fuse.

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Because the z-axis thickness of our optical section was appreciable on the scale of our x-y resolution (see Materials and methods), one concern is that curvature of the plasma membrane in the z-axis could make fusions that actually occurred at the membrane near the top or bottom of the optical section appear to be nearer to the center of the ribbon. However, the majority of pHluorin events still arose at a substantial distance from the estimated membrane position, even after correcting for z-axis curvature (dashed arrow, Figure 5E). Another source of uncertainty in localizing pHluorin events could be changes in ribbon position along the z-axis within the optical section, since we normalized x-axis positions of pHluorin events with respect to the center of the ribbon in constructing the histogram shown in Figure 5E. Major errors seem unlikely to arise from such changes in ribbon position within the optical section, because light from the entire optical section contributes to the x-axis intensity profile used to localize the ribbon in a given focal position. To estimate the extent of error from this source, we measured the change in x-axis position of ribbons as the focal plane was varied within a z-axis range from appearance to disappearance of the ribbon. As shown in Figure 5—figure supplement 2, the center of the Gaussian component of the fit to the RBP profile varied somewhat with focal position, especially near the upper and lower z-axis bounds, where fluorescence intensity of the ribbon was lower (e.g., sample traces in Figure 5—figure supplement 2A,B). However, the estimated x-axis position of the ribbon varied by less than ± 50 nm throughout the central part of the sampled z-axis range (Figure 5—figure supplement 2C). Therefore, the observed uncertainty in the x-axis position of ribbons within the optical section is insufficient to account for the wide range of pHluorin-event locations (Figure 5E).

We conclude that pHluorin events associated with ribbons arose both near the membrane and at more distal locations, at a substantial distance from the membrane. A simple explanation is that the distal pHluorin events represent fusion of synaptic vesicles with other ribbon-attached vesicles, which have themselves already undergone exocytosis and provide a path to the extracellular space. This model is also consistent with previous work using electron microscopy (Matthews and Sterling, 2008), which suggested the existence of such vesicle-vesicle compound fusion at ribbon synapses of goldfish BPCs.

In this study, we exploited the special features of ribbon synapses of retinal BPCs to observe the trafficking of single synaptic vesicles associated with the CAZ during ongoing transmitter release. Prior studies of synaptic vesicle trafficking at BPC ribbon synapses using TIRFM focused on a narrow region extending ~100 nm from the plasma membrane and revealed vesicle movements within this region, as well as vesicle exocytosis at the membrane (Zenisek et al., 2000, 2002; Holt et al., 2004; Midorikawa et al., 2007; Zenisek, 2008). By contrast, our imaging approaches allowed vesicle motion and fusion to be detected across the entire population of ribbon-associated vesicles, albeit at a spatial resolution insufficient for detection of small movements of less than a vesicle diameter, like those reported in TIRFM experiments (Zenisek et al., 2000). Therefore, our studies target a different set of questions and provide results that are complementary to the previous studies of synaptic vesicle dynamics at ribbon active zones.

Our results suggest a dual role for the synaptic ribbon in neurotransmitter release at the active zone, with the ribbon serving as both a conduit for diffusion of tethered synaptic vesicles and a platform for vesicles to fuse distal to the plasma membrane. A likely mechanism for such distal fusions is compound fusion of synaptic vesicles with other vesicles. Diffusion appeared to dominate for ribbon-associated vesicles farthest from the plasma membrane, which moved toward the center of the ribbon prior to loss during sustained depolarization. Synaptic vesicle fusion detected by pHluorin events occurred most frequently on the membrane-proximal half of the ribbon, perhaps because calcium levels are higher nearer the membrane during depolarization (Zenisek et al., 2003; Francis et al., 2011; Vaithianathan and Matthews, 2014). These fusion events include some that likely occurred directly at the plasma membrane, which possibly correspond to the fusions seen in TIRFM experiments. However, many pHluorin events arose at distances >100 nm from the estimated position of the plasma membrane, suggesting that compound fusion contributes significantly to ongoing transmitter release at ribbon synapses during sustained depolarization. Overall, our results set the stage for models of synaptic vesicle trafficking and fusion at the ribbon active zone that incorporate calcium signaling, the kinetics of tethered diffusion of vesicles, and vesicle-vesicle fusion at a distance from the plasma membrane.

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Author details

1. Thirumalini Vaithianathan

   Department of Neurobiology and Behavior, Stony Brook University, New York, United States

   Contribution

   TV, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Diane Henry

   Department of Neurobiology and Behavior, Stony Brook University, New York, United States

   Contribution

   DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Wendy Akmentin

   Department of Neurobiology and Behavior, Stony Brook University, New York, United States

   Contribution

   WA, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Gary Matthews

   1. Department of Neurobiology and Behavior, Stony Brook University, New York, United States
   2. Department of Ophthalmology, Stony Brook University, New York, United States

   Contribution

   GM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   Gary.G.Matthews@stonybrook.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-7915-9126

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