Bruchpilot and Synaptotagmin collaborate to drive rapid glutamate release and active zone differentiation

The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.


INTRODUCTION
Active zones (AZs) allow exquisite spatial and temporal control of vesicle fusion. Large multidomain proteins rich in coiled-coil sequences such as Bassoon, Piccolo and the CAST/ERC family member Brp are major structural and functional organizers of AZs (Südhof, 2012). Their abundance appears to correlate positively with neurotransmitter release (Graf et al., 2009;Matz et al., 2010;Weyhersmüller et al., 2011;Ehmann et al., 2014;Peled et al., 2014).
At Drosophila melanogaster NMJs, Brp is crucial for synchronous glutamate release and the clustering of calcium channels at AZs (Kittel et al., 2006;Wagh et al., 2006). Linking the amount of Brp or Bassoon per AZ to the number and spatial arrangement of calcium channels may account for the correlation with release probability, e.g., in the context of synaptic homeostasis (Matz et al., 2010;Weyhersmüller et al., 2011;Ehmann et al., 2014). Slight increases in coupling distance in the 20-40 nm range reduce release probability dramatically while changing kinetic release parameters to a lesser extent (Neher, 1998;Eggermann et al., 2011;Schmidt et al., 2013;Vyleta and Jonas, 2014). Differences in coupling distance are therefore ideal for scaling the amount of release, whereas controlling its time course appears to require additional molecular mechanisms.
The main kinetic transmitter release parameters are synaptic delay, rise, and decay times. In the present study, we focus on synaptic delay and EPSC rise time. Notably, the latter is increased by more than 1 ms at synapses lacking Brp, while release probability drops by comparison only moderately (Kittel et al., 2006;Eggermann et al., 2011). This marked kinetic change appears disproportional to the reduction in release probability. While synaptic delay has not yet been analyzed at brp 69 synapses, it is usually fairly constant for a wide range of release probabilities (Barrett and Stevens, 1972;Datyner and Gage, 1980). While the molecular mechanisms controlling release kinetics are complex and not well understood (Neher, 2010), it is Frontiers in Cellular Neuroscience www.frontiersin.org clear that the vesicle protein Syt plays an important role (Brose et al., 2002;Young and Neher, 2009). As initially suggested more than 20 years ago (DiAntonio et al., 1993;Littleton et al., 1993;Geppert et al., 1994), Syt is crucial for triggering release and may act both as a calcium sensor and a vesicle fusion clamp.
In fact, its role may change from clamp to sensor upon calcium influx into the presynaptic terminal (DeBello et al., 1993;Walter et al., 2011).
To clarify the molecular mechanisms that shape the time course of release we analyzed the interaction between Brp and Syt. We find that in addition to prolonged EPSC rise time, synaptic delay is strongly increased at brp 69 synapses. Interestingly, whereas Syt is necessary for the increase in both kinetic parameters, it has little effect on the amount of transmitter released from brp 69 AZs. Following up on the functional interaction of Brp and Syt, our data suggest central roles of these two proteins in the spatial differentiation of AZs and reveal that the number of AZs per bouton, as well as the number and distribution of Brp molecules per AZ is Syt-dependent.

PHYSIOLOGICAL SOLUTION AND PREPARATION
The composition of the extracellular, physiological, hemolymphlike saline (HL-3, Stewart et al., 1994) was (in mM): NaCl 70, KCl 5, MgCl 2 20, NaHCO 3 10, trehalose 5, sucrose 115, HEPES 5, CaCl 2 as indicated, pH adjusted to 7.2. Wandering male third instar larvae were dissected in HL-3 without CaCl 2 . All experiments were carried out at NMJs formed on ventral abdominal muscles 6/7 in segments A2 and A3. Figure 1 were performed in HL-3 saline containing 1 mM [Ca 2+ ] Ex essentially as reported previously (Pawlu et al., 2004). Bath temperature was kept constant at 18 ± 0.5 • C using a Peltier element (27 W, Conrad Electronic) glued to the bath inflow with heat-conductive paste (Fischer Elektronik). EPSCs were elicited using a 0.2 Hz nerve-stimulation protocol with 0.2 ms pulse duration and amplitudes slightly above the threshold for eliciting an action potential via a suction electrode (filled with extracellular solution). Recording electrodes with openings of about 5-10 μm diameter below the tip had resistances of 250 k when filled with HL-3. About 20 EPSCs were recorded per site and analyzed with IgorPro 5.04 (Wavemetrics). The data were digitally filtered at 3 kHz (Gaussian filter), baseline subtracted and the average of all failures was FIGURE 1 | Presynaptic action potential and synaptic delay in brp 69 . (A) Focal recording from a Drosophila wt NMJ. Nerve stimulation (left arrow) elicited an action potential in the suction electrode which traveled to the presynaptic bouton under the focal electrode and lead to deflections (presynaptic AP, second arrow) prior to the large compound EPSC (third arrow). A subthreshold pulse through the focal electrode elicited the final upward deflection (right side of the trace and allowed to measure electrode and seal resistance). The lower panel shows the enlarged presynaptic AP and illustrates how half-duration of the positive AP deflection (red) and synaptic delay (blue) were determined. (B) While AP half-duration is unchanged between brp 69 (green) and wt (black), (C) synaptic delay is prolonged in brp 69 . Shown are single values (dots) and mean ± SD.

Frontiers in Cellular Neuroscience
www.frontiersin.org subtracted from the currents. AP durations were measured at half amplitude of the positive deflection (Dudel, 1965) and synaptic delay was measured from the peak of the AP to the point at which the back extrapolation of the EPSC current rising phase crossed the baseline (Figure 1). Focal recordings in Figure 6 were performed in HL-3 saline containing 0.5 mM [Ca 2+ ] Ex . Bath temperature was kept constant at 20 ± 1 • C. Focal electrodes (resistances 600 ± 50 k when filled with HL-3 solution) were positioned on proximal or distal type Ib boutons of muscles 6/7. EPSCs were elicited using a 0.2 Hz nerve-stimulation protocol with 0.2 ms pulse duration and 7 V amplitude. Traces were low-pass filtered at 20 kHz, and recorded and stored with Patchmaster using an EPC10 double patch clamp amplifier (HEKA electronics). About 60 EPSCs were averaged per site and analyzed with Igor Pro 6.05 (Wavemetrics). 10 mg EGTA-AM (membrane permeable tetraacetoxymethyl ester of ehtyleneglycol-bis(ßaminoethyl)-N,N,N' ,N'-tetraacetic acid, Calbiochem Germany) was dissolved in DMSO with 20% Pluronic (Invitrogen) to obtain a stock solution of 10 mM EGTA. This stock solution was diluted 1:100 with calcium-free HL-3 and applied to the dissected preparation for 10 min. After incubation preparations were washed for 5 minutes with HL-3 (Müller et al., 2012) and recordings were performed in HL-3 containing 1.0 mM [Ca 2+ ] Ex as described above.

TWO-ELECTRODE VOLTAGE CLAMP RECORDINGS (TEVC)
Two-electrode voltage clamp-recordings (Figure 3) were performed essentially as previously described (Kittel et al., 2006) using an Axo Clamp 2B amplifier (Axon Instruments, Molecular Devices). All measurements were made from muscle 6 at 21 ± 1 • C bath temperature. Intracellular electrodes were filled with 3 M KCl and had resistances of 12-15 M . V holding was −60 mV for evoked EPSCs. Only cells with an initial membrane potential of at least −50 mV and ≥4 M input resistance were analyzed. Synaptic responses were generated by pulses of 0.3 ms length and 5-10 V amplitude, applied via a suction electrode (filled with extracellular solution) and low-pass filtered at 10 kHz. We applied a 0.2 Hz stimulation protocol, averaged 20 EPSCs per muscle cell and analyzed the data with Clampfit (Axon Instruments, Molecular Devices).

IMAGING
Larvae were dissected in ice-cold HL-3 standard saline without CaCl 2 , fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 10 min and blocked with PBT (PBS containing 0.05% Triton X-100, Sigma) including 5% natural goat serum (Dianova) for 30 min. Primary antibodies were added for overnight staining at 4 • C. After three washing steps with PBS (20 min each), preparations were incubated with secondary antibodies for 2-4 h at room temperature followed by three washing steps with PBS. Filets were mounted using Vectashield (Vector Laboratories) and images were aquired using an Apotome System (Zeiss, Axiovert 200M Zeiss, objective 63x, NA 1.4, oil). Antibodies were used at the following concentrations: mouse monoclonal antibody (mAb) Brp Nc82 (1:250), Alexa Fluor 488-conjugated goat α-mouse (Invitrogen) and Cy3-conjugated goat α-horseradish peroxidase (HRP, Jackson Immuno Research) antibodies (1:250), rabbit α-Dsyt-CL1 (Mackler et al., 2002) and Cy3-conjugated goat α-rabbit (Jackson Immuno Research, 1:250) antibodies. Z-stacks of 15-20 single images taken every 250 nm were maximum projected and analyzed in ImageJ (1.440, NIH). Brp puncta per NMJ and per bouton were quantified manually. Using the three terminal boutons of type Ib and Is branches the respective structural gradient was analyzed. Distal boutons were located at the end of bouton chains, while proximal boutons were closer to the entry site of the motor neuron. Bouton area, length (along chain axis) and width (90 • to length) were measured using α-HRP stainings.
Frontiers in Cellular Neuroscience www.frontiersin.org Localization densities were analyzed only in boutons with areas <10 μm.

STATISTICAL ANALYSIS
Statistical analyses were performed with Sigma Plot 12 (Systat Software) using the non-parametric Mann-Whitney rank sum test. Linear fits for mean ± SEM Brp localizations per AZ (Figure 9) were made in Igor Pro (Wavemetrics) and statistical analysis was performed using the non-parametric Spearman correlation coefficient. Asterisks indicate the significance level ( * p < 0.05, * * p < 0.01, * * * p < 0.001). Data are reported as mean ± SEM unless indicated otherwise and n denotes sample number.

SYNAPTIC DELAY IS INCREASED IN brp 69
We performed focal recordings using macropatch electrodes which allow simultaneous measurements of presynaptic action potentials (AP) and synaptic release (Dudel, 1965). EPSCs evoked by 0.2 Hz nerve stimulation were recorded at brp 69 and wt larval NMJs on muscles 6/7 to measure half-duration of the positive AP deflection and synaptic delay ( Figure 1A). Whereas AP wave form was unchanged in brp 69 compared to wt, synaptic delay was significantly increased (2.27 ± 0.3 ms and 1.8 ± 0.3 ms mean ± SD, p = 0.0014, n = 14, and 13 for brp 69 and wt, Figures 1B,C).
As quantal time course is normal in brp 69 (Kittel et al., 2006), Frontiers in Cellular Neuroscience www.frontiersin.org the increase in release kinetics at brp 69 synapses is likely due to alterations in presynaptic fusion mechanisms.

IMPACT OF SYT ON DIFFERENT AZ STATES
We used wt, brp 69 and rab3 rup to define explicit AZ conditions: (i) normal organization, (ii) disorganized lacking Brp (Kittel et al., 2006), and (iii) large accumulation of Brp proteins (Graf et al., 2009;Ehmann et al., 2014), respectively. To determine the impact of the putative calcium sensor Syt on synchronous transmitter release in the context of different AZ states we combined these genotypes with syt KD . Protein levels of endogeneous Syt were decreased via RNAi (syt1-RNAi 8875 , see experimental procedures). By engaging the binary UAS-Gal4 expression system (Brand and Perrimon, 1993), syt1-RNAi was driven in larval glutamatergic motor neurons or panneuronally. To confirm that presynaptic Syt expression was reduced by this strategy, immunostainings of larval NMJs were performed using an antiserum against Syt1 (Mackler et al., 2002; Figure 2). Whereas presynaptic terminals of syt AD4 were completely devoid of Syt, there was residual though heavily reduced protein expression in syt KD compared to wt ( Figure 2C). Furthermore, we quantified the protein reduction following Syt knock-down with dSTORM (Ehmann et al., 2014). Comparison of Syt1 localization numbers in boutons of similar size in wt and syt KD revealed a reduction to 62.38% in syt KD (Figures 2D-F). To address the functional consequences of syt KD at wt, brp 69 , and rab3 rup synapses, postsynaptic currents in response to low-frequency nerve stimulation were recorded in two-electrode voltage clamp mode (TEVC) from larval ventral abdominal muscles 6/7 (Figure 3). Both panneuronal (Figure 3) and motoneuronal (data not shown) RNAi expression gave essentially comparable results. At wt synapses, syt KD decreased EPSC amplitude and lengthened rise time (amplitude: 30.0 ± 4.5 nA and 53.6 ± 4.7 nA, p = 0.002; rt: 1.3 ± 0.1 ms and 1.0 ± 0.03 ms, p < 0.001; n = 11 and 17 for syt KD and wt, Figure 3B) consistent with the role of Syt as a sensor for fast release (DiAntonio et al., 1993;Littleton et al., 1993). Similarly, at rab3 rup synapses, syt KD reduced the amplitude and increased the delay of postsynaptic responses (amplitude: 19.5 ± 2.1 nA and 50.3 ± 5.3 nA, p < 0.001; delay: 1.7 ± 0.1 ms and 1.4 ± 0.04 ms, p = 0.013; n = 14 and 10 for rab3 rup , syt KD , and rab3 rup ). Strikingly, syt KD at brp 69 synapses left current amplitudes unchanged (35.1 ± 6.7 nA and 24.8 ± 3.3 nA, p > 0.05) and in fact accelerated EPSC rise time and delay (rt: 1.4 ± 0.1 ms and 2.2 ± 0.2 ms, p = 0.008; delay: 1.6 ± 0.03 ms and 1.9 ± 0.1 ms, p < 0.001; n = 12 and 11 for brp 69 , syt KD , and brp 69 ). These results illustrate that Syt is necessary for efficient and rapid vesicle fusion at AZs with normal or increased Brp levels. In contrast, vesicle release from brp 69 AZs appears less dependent on Syt. We did not find any changes in size or kinetics of quantal events in brp 69 and brp 69 , syt KD that could explain these effects (amplitude: 0.89 ± 0.04 nA and 0.90 ± 0.04 nA; rt: 1.0 ± 0.06 ms and 1.0 ± 0.04 ms; tau: 6.02 ± 0.6 ms and 7.2 ± 0.4 ms; all p > 0.05; n = 10 and 14 for brp 69 and brp 69 , syt K D , respectively). Thus, the changes in release kinetics following syt KD suggest that Syt protracts release at brp 69 AZs.

REDUCED EGTA SENSITIVITY IN brp 69 , syt KD
To further clarify how syt KD affects release we tested the influence of EGTA-AM in syt KD and brp 69 , syt KD in focal recordings Frontiers in Cellular Neuroscience www.frontiersin.org (Figure 4, Kittel et al., 2006). Consistent with earlier work (Maximov and Südhof, 2005) release in syt KD was significantly reduced (0.37 ± 0.03 nA and 0.22 ± 0.03 nA, p = 0.002, n = 16 each), whereas the reduction was not significant in brp 69 , syt KD (0.28 ± 0.05 nA and 0.21 ± 0.03 nA, p > 0.05, n = 17, and 10 without and with EGTA). This is in contrast to the findings described in Kittel et al. (2006) for brp 69 and suggests that Syt knock-down reduces coupling distance in brp 69 and that Syt's role in positional priming (Young and Neher, 2009) requires Brp.

SYT AND BRP ARE ESSENTIAL FOR FUNCTIONAL PRESYNAPTIC DIFFERENTIATION
We used focal electrodes as these can be selectively placed on a subset of presynaptic boutons to improve spatial resolution of synaptic measurements. Boutons were visualized by GFP-expression (Pawlu et al., 2004) and postsynaptic currents of proximal and distal type Ib boutons were measured in response to low-frequency nerve stimulation in 0.5 mM [Ca 2+ ] Ex (Figure 6). Distal boutons of wt NMJs showed larger EPSC amplitudes and shorter rise times than proximal boutons (amplitude: 1.4 ± 0.1 nA and 1.0 ± 0.1 nA, p = 0.005; rt: 1.1 ± 0.07 ms and 1.3 ± 0.07 ms, p = 0.032, n = 24, and 33, Figure 6B). In contrast, at both brp 69 and syt KD NMJs amplitude and kinetics of postsynaptic currents were comparable in distal and proximal boutons (n = 10 and 11 for brp 69 and 11 and 13 for syt KD , Figure 6B). These results reveal that Brp and Syt are both essential for the functional differentiation of the NMJ.

SYT GUIDES AZ DISTRIBUTION
To further investigate the impact of Brp and Syt on maintaining the structural gradient we again performed immunostainings (Figure 8). At brp 69 NMJs, bouton area, length, and width were larger for distal than for proximal type Ib boutons (data not shown). Analysis of syt KD NMJs showed profound alterations of synaptic morphology regarding Brp distribution and bouton size. Whereas the number of Brp positive AZs per NMJ was slightly decreased compared to wt (724 ± 38 and 861 ± 41, p = 0.019, n = 16 NMJs each), AZ numbers per Ib bouton were reduced to about a quarter. Furthermore, Brp was distributed homogeneously along the MN6/7b-Ib motor neuron and spatial dimensions of type Ib boutons were similar for all locations along the motoneuron [p > 0.05, n = 125 (1), 109 (2), 109 (3), Figures 8B,C]. Compared to wt, bouton size was reduced dramatically. In addition, analysis of the structural gradient in combined rab3 rup , syt KD animals revealed that Syt knock-down also decreases the structural differentation at rab3 rup NMJs (data not shown). These data demonstrate that Syt is essential for the structural differentation of the NMJ both in wt and rab3 rup .

SYT INFLUENCES ORGANIZATION AND NUMBER OF BRP PROTEINS AT INDIVIDUAL AZs
In a final set of experiments we employed dSTORM to image glutamatergic boutons. This localization microscopy technique substantially increases spatial resolution compared to conventional Frontiers in Cellular Neuroscience www.frontiersin.org fluorescence light microscopy van de Linde et al., 2011) and can provide quantitative insight into the nanoscopic organization of presynaptic AZs (Sauer, 2013;Ehmann et al., 2014Ehmann et al., , 2015. dSTORM resolved the substructural arrangement of indiviudal Brp localizations into multiple clusters within single AZs, which correspond to diffraction-limited Brp puncta in confocal images. We analyzed AZs in the distal six type Ib boutons of MN6/7b-Ib motor neurons in wt and syt KD (Figure 9). Interestingly, syt KD AZs were larger than their wt counterparts (0.079 ± 0.003 μm 2 and 0.069 ± 0.002 μm 2 , p = 0.003, n = 300, and 468 AZs, Figure 9D) and contained more localizations (710 ± 30 and 590 ± 19, p = 0.003, Figure 9E), which reflects an increased number of Brp protein copies (Ehmann et al., 2014). Furthermore, we observed that syt KD AZs contained a similar number of Brp localizations irrespective of bouton order ( Figure 9F, Spearman correlation coefficient r = −0.169, p < 0.001 for wt and r = 0.014, p > 0.05 for syt KD indicates a moderate negative correlation for wt and no correlation for syt KD ). Thus, Syt influences the arrangement of Brp at the AZ. Previous work showed that the number of Brp localizations per wt AZ is higher in distal than in proximal type Ib boutons (Ehmann et al., 2014). This is consistent with the electrophysiological and structural data presented here.

DISCUSSION
While differentiation of presynaptic terminals was initially described more than 50 years ago and has since been studied extensively in various organisms, its mechanisms remain poorly understood (Katz, 1936;Hoyle and Wiersma, 1958;Reyes et al., 1998;reviewed in Atwood and Karunanithi, 2002). In view of the enormous complexity of the relevant molecular mechanisms (Südhof, 2012), the genetically and experimentally accessible NMJ of Drosophila melanogaster provides advantageous features for studying a glutamatergic synaptic system. Type Is-and Ib-boutons of the NMJ exhibit distinct functional properties (Kurdyak et al., 1994;Pawlu et al., 2004), show differences in vesicle size  and in the amount of Brp molecules per AZ (Ehmann et al., 2014).
Here we show branch-specific differentiation in the MN6/7b-Ib motoneuron regarding structure and function. Distal type Ib boutons are larger than proximal ones, have more Brp positive AZs and show larger and faster postsynaptic responses (Figures 5 and 6). Consistent with these findings, AZs of distal type Ib boutons are larger and possess more Brp molecules per AZ (Ehmann et al., 2014). Presynaptic differentiation is impaired by disrupting either Brp or Syt function (Figures 6, 8, and 9). Postsynaptic responses of proximal and distal boutons in brp 69 and syt KD are comparable and the structural gradient in bouton size, AZs per bouton and AZ size is absent in syt KD . Moreover, genetic evidence suggests that Brp and Syt act in the same functional pathway to mediate structural heterogeneity (Figure 7). Structural and functional presynaptic differentiation thus clearly requires the concerted action of Brp and Syt.
Interestingly, we obtained consistently lower values for AZ size and Brp counts per AZ than a recent previous investigation using dSTORM (Ehmann et al., 2014). In the present study, we raised both mutant and control animals at 29 • C to ensure efficient RNA-mediated syt KD (expression via the GAL4-UAS system is temperature-dependent), whereas Ehmann et al. (2014) raised larvae at 25 • C. Since higher temperature accelerates the development of Drosophila, enlarges presynaptic arborizations and increases the number of AZs per NMJ (Ashburner, 1989;Sigrist et al., 2003), it is conceivable that temperaturedependent plasticity also affects molecular organization at the level of individual AZs.
The molecular mechanisms controlling size, structure and distribution of AZs are complex. Several years ago, the vesicle protein Rab3 was identified as an important regulatory factor of AZ size and distribution (Graf et al., 2009). Rab-proteins Frontiers in Cellular Neuroscience www.frontiersin.org are key organizers of vesicle trafficking (Harris and Littleton, 2011). Measurements with genetically encoded postsynaptic calcium sensor showed comparable calcium signals in proximal and distal type Ib boutons at rab3 rup NMJs (Peled and Isacoff, 2011). Here, we found a gradient in rab3 rup animals in the number of Brp positive AZs along the MN6/7b-Ib motoneuron (Figure 5), unlike in syt KD (Figure 8). Whereas at rab3 rup NMJs the overall number of AZs is reduced dramatically (Graf et al., 2009), this reduction is moderate at syt KD NMJs (724 ± 38 and 861 ± 41, see Results). However, at both rab3 rup and syt KD type Ib branches, the number of Brp proteins per AZ is increased strongly and moderately, respectively (Ehmann et al., 2014; Figure 9). In contrast to rab3 rup synapses, syt KD decreases bouton size. Smaller boutons were reported for syt AD4 and linked to defects in endocytosis (Dickman et al., 2006). We found that brp and syt interact genetically regarding AZ number per bouton, but not area of boutons (Figure 7), which suggests that the effects of Syt on bouton size and AZ-differentiation are not strictly linked.
The pronounced presynaptic structural alterations after syt KD are puzzling. Syt is one of the best-studied synaptic proteins. However, its function has mainly been discussed without considering AZ-differentiation. After syt KD , evoked release was reduced by a factor of 4-5 compared to wt in our focal recordings (Figure 6). However, the number of Brp spots in terminal boutons was also reduced by a factor of 4-5 after syt KD (Figure 8). Is release probability per AZ in distal boutons following syt KD therefore similar to wt? More work perhaps combining optical release sensors, focal recordings and subsequent immunostainings will be necessary to clarify this issue. The present study highlights how interpretations of synaptic function and differentiation profit from electrophysiological recording techniques with improved spatial resolution (focal vs. TEVC). Functional sampling of synaptic subsets appears absolutely necessary when considering the significant differentiation at the structural level. Either way, linking structure and function at one and the same AZ is fundamentally important for a comprehensive mechanistic interpretation (Bailey and Chen, 1983;Wojtowicz et al., 1994).
Our electrophysiological data suggest that Syt protracts release at AZs lacking Brp. In this study, we used syt KD to reduce the protein level in presynpatic terminals (Figure 2). While we assume there are normally more than 10 Syt molecules on each vesicle in our preparation (Takamori et al., 2006), it is unclear whether syt KD leads to a reduction in the average number of Syt proteins per vesicle or a reduction in the number of Syt positive vesicles with those remaining possessing a full complement of Syt copies. This is relevant in the context of the molecular interpretation of our results. For example, ring-like oligomerization of Syt's cytosolic C2-domains, which prevents release in the absence of calcium, requires a certain copy number (Wang et al., 2014). Furthermore, quantitative information on Syt's partner molecules, such as Complexin and SNAREproteins, will be required for a mechanistic interpretation down to the level of stochiometric interactions (Mohrmann et al., 2010;Cho et al., 2014). Imaging techniques such as dSTORM can be used to quantify the molecular organization of AZs (Sauer, 2013;Ehmann et al., 2015) and will be necessary to Frontiers in Cellular Neuroscience www.frontiersin.org clarify the so far insufficiently understood kinetic release parameters. In this context, interpretations may well have to take into account the existence of alternative sensors (Walter et al., 2011). Syt contributes to vesicle docking at the AZ, vesicle positioning within the AZ, clamping and triggering release from the AZ (Walter et al., 2011). While specific amino acids of Syt have been put in connection with certain subsets of these features (e.g., Young and Neher, 2009), it remains unclear which specific functional roles and molecular domains of Syt are responsible for interactions with Brp and structural synaptic specialization. Intriguingly, both Syt and Rab3 are involved in vesicle trafficking and participate in the structural differentiation of AZs. Our work supports the notion that organization of the synaptic vesicle cycle and AZ structure are causally linked.