Drosophila larvae were raised in vials on standard corn meal (Ashburner, 1989) at 25°C for focal recordings in Figure 1 (wt and brp⁶⁹) or at 29°C for reliable RNAi expression in all other experiments (mutant and control groups). For Syt RNAi, we expressed syt1_RNAi⁸⁸⁷⁵ Vienna Drosophila Resource Center (VDRC) specifically in motor neurons (ok6-GAL4/+; UAS-syt1_RNAi⁸⁸⁷⁵/+) or panneuronally (elav-GAL4/UAS-syt1_RNAi⁸⁸⁷⁵) using the binary UAS-GAL4 expression system (Brand and Perrimon, 1993). brp⁶⁹ and rab3^rup were used as previously described (Kittel et al., 2006; Graf et al., 2009). To combine brp⁶⁹ and rab3^rup with syt1_RNAi⁸⁸⁷⁵ the following lines were generated: brp⁶⁹ok6-GAL4/ df(2R)BSC29; UAS-syt1_RNAi⁸⁸⁷⁵/+ and brp⁶⁹/df(2R)BSC29; elav-GAL4/UAS-syt1_RNAi⁸⁸⁷⁵ (brp⁶⁹, syt^KD); rab3^rup/df(2R)ED2076 ok6-GAL4; UAS-syt1_RNAi⁸⁸⁷⁵/+ and rab3^rup/df(2R)ED2076; elav-GAL4/UAS-syt1_RNAi⁸⁸⁷⁵ (rab3^rup, syt^KD); ok6-GAL4/+ and elav-GAL4/+ served as wt controls. For focal recordings, GFP was expressed in presynaptic terminals: ok6-GAL4/+; UAS-CD8::GFP/+ (wt), brp⁶⁹ok6-GAL4/df(2R)BSC29;UAS-CD8::GFP/+ (brp⁶⁹) and ok6-GAL4/+; UAS-CD8::GFP/UAS-Syt1_RNAi⁸⁸⁷⁵ (syt^KD). For non-allelic non-complementation the following genotypes were used: w¹¹¹⁸ (wt), syt1^AD4/+ (DiAntonio et al., 1993), brp⁶⁹/+ (Kittel et al., 2006) and syt1^AD4/ brp⁶⁹ (syt^AD4+/+brp⁶⁹).

The composition of the extracellular, physiological, hemolymph-like saline (HL-3, Stewart et al., 1994) was (in mM): NaCl 70, KCl 5, MgCl₂ 20, NaHCO₃ 10, trehalose 5, sucrose 115, HEPES 5, CaCl₂ as indicated, pH adjusted to 7.2. Wandering male third instar larvae were dissected in HL-3 without CaCl₂. All experiments were carried out at NMJs formed on ventral abdominal muscles 6/7 in segments A2 and A3.

Macropatch recordings in Figure 1 were performed in HL-3 saline containing 1 mM [Ca²⁺]_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 kΩ 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 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 [Ca²⁺]_Ex. Bath temperature was kept constant at 20 ± 1°C. Focal electrodes (resistances 600 ± 50 kΩ 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(ß-aminoethyl)-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 [Ca²⁺]_Ex as described above.

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 MΩ. V_holding was -60 mV for evoked EPSCs. Only cells with an initial membrane potential of at least -50 mV and ≥4 MΩ 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).

Larvae were dissected in ice-cold HL-3 standard saline without CaCl₂, 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.

Larvae were dissected, fixed and washed as described above and super-resolution imaging was performed essentially as previously reported (Ehmann et al., 2014). Preparations were incubated with mAb Brp^Nc82 (1:2000) and secondary antibody goat α-mouse F(ab’)₂ fragments (A10534, Invitrogen) labeled with Cy5-NHS (PA15101, GE Healthcare) at a concentration of 5.2 × 10^-8 M or with rabbit α-Dsyt-CL1 (1:10000, Mackler et al., 2002) and secondary antibody goat α-rabbit F(ab’)₂ fragments labeled with Alexa Fluor 647 (1:500, Invitrogen). Boutons were visualized with Alexa Fluor 488 conjugated goat α-horseradish-peroxidase antibody (α-HRP, 1:250, Jackson Immuno Research). After staining, larval preparations were incubated in 100 mM Mercaptoethylamin (MEA, Sigma-Aldrich) buffer in PBS, pH 7.8–7.9 to allow reversible switching of single fluorophores during data acquisiton (van de Linde et al., 2008). Images were acquired using an inverted microscope (Olympus IX-71, 60x, NA 1.45, oil immersion) equiped with a nosepiece-stage (IX2-NPS, Olympus). 644 nm (iBEAM-SMART-640-S, Toptica), and 488 nm (iBEAM-SMART-488-S, Toptica) lasers were used for excitation of Cy5 and Alexa Fluor488, respectively. Laser beams were passed through a clean-up filter (Brightline HC 642/10, Semrock, and ZET 488/10, Chroma, respectively) and two dichroic mirrors (Laser-MUX BS 514-543 and HC-quadband BP, Semrock) onto the probe. The emmitted fluorescence was filtered with a quadband-filter (HC-quadband 446/523/600/677, Semrock) and divided onto two cameras (iXon Ultra DU-897-U, Andor) using a dichroic mirror (HC-BS 640 imaging, Semrock). In addition, fluorescence was filtered using a longpass- (Edge Basic 635, Semrock) or bandpass-filter (Brightline HC 525/50, Semrock) for red and green channels, respectively. Pixel sizes were 126 nm (red) and 128 nm (green). Single fluorophores were localized and high resolution-images were reconstructed with rapidSTORM (Heilemann et al., 2008; Wolter et al., 2010; van de Linde et al., 2011; Wolter et al., 2012; www.super-resolution.de). Only fluorescence spots with more than 1000 photons were analyzed (10 nm /pixel sub-pixel binning). Data were processed with ImageJ (1.440, NIH), AZ area and Brp localizations per AZ were analyzed as previously described Ehmann et al. (2014). AZs were assigned to single boutons using the α-HRP signal. For Syt1 quantification (Figure 2) we determined localization counts in single type Ib boutons that were defined according to the α-HRP signal. Localization densities were analyzed only in boutons with areas <10 μm.

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.

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 brp⁶⁹ 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 brp⁶⁹ 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 brp⁶⁹ and wt, Figures 1B,C). As quantal time course is normal in brp⁶⁹ (Kittel et al., 2006), the increase in release kinetics at brp⁶⁹ synapses is likely due to alterations in presynaptic fusion mechanisms.

We used wt, brp⁶⁹ 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-RNAi⁸⁸⁷⁵, 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, brp⁶⁹, 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 brp⁶⁹ 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 brp⁶⁹, syt^KD, and brp⁶⁹). 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 brp⁶⁹ AZs appears less dependent on Syt. We did not find any changes in size or kinetics of quantal events in brp⁶⁹ and brp⁶⁹, 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 brp⁶⁹ and brp⁶⁹, syt^KD, respectively). Thus, the changes in release kinetics following syt^KD suggest that Syt protracts release at brp⁶⁹ AZs.

To further clarify how syt^KD affects release we tested the influence of EGTA-AM in syt^KD and brp⁶⁹, syt^KD in focal recordings (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 brp⁶⁹, 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 brp⁶⁹ and suggests that Syt knock-down reduces coupling distance in brp⁶⁹ and that Syt’s role in positional priming (Young and Neher, 2009) requires Brp.

The innervation of ventral abdominal muscles 6/7 is shared between two functionally distinct motoneurons in Drosophila larvae (Karunanithi et al., 2002): the MN6/7b-Ib (RP3) neuron gives rise to large type Ib boutons (Atwood et al., 1993), whereas the MNSNb/d-Is neuron forms smaller type Is boutons (Kurdyak et al., 1994; Lnenicka and Keshishian, 2000; Hoang and Chiba, 2001). We performed immunostainings and counted the number of Brp puncta per bouton as an estimate of the number of AZs. In addition, staining against horseradish-peroxidase (HRP) was used to measure dimensions of presynaptic arborizations (Jan and Jan, 1982; Figure 5). We found an uneven Brp distribution in wt type Ib motoneurons with more Brp positive AZs in distal than in proximal boutons (16.5 ± 0.8, 9.8 ± 0.7, 9.6 ± 0.8, p < 0.001, n = 115 (1), 70 (2), 70 (3), Figure 5B). In addition, distal type Ib boutons were largest along the bouton chain (10.6 ± 0.5 μm², 6.9 ± 0.5 μm², 6.5 ± 0.5 μm², p < 0.001). Interestingly, this gradient was not found in type Is boutons regarding both AZ number [5.5 ± 0.4, 5.5 ± 0.3, 4.6 ± 0.3, p > 0.05, n = 73 (1), 73 (2), 73 (3)] and bouton area (3.0 ± 0.2 μm², 3.1 ± 0.2 μm², 3.3 ± 0.2 μm², p > 0.05, Figure 5C). Our data are in line with previous work analyzing functional (Guerrero et al., 2005; Peled and Isacoff, 2011) and structural properties (Ehmann et al., 2014) of the NMJ. Furthermore, the number of AZs per bouton was decreased at rab3^rup NMJs compared to wt (Figures 5D–F). This matches earlier work, showing that Rab3 controls the distribution of Brp at the NMJ with decreased AZ numbers per NMJ and increased Brp levels per AZ in rab3^rup (Graf et al., 2009). However, the structural gradient regarding AZ number per bouton and bouton size was still present in rab3^rup type Ib axons [Brp: 7.0 ± 0.3, 4.1 ± 0.3, 4.2 ± 0.3; area: 10.5 ± 0.5 μm², 6.9 ± 0.5 μm², 7.5 ± 0.5 μm², p < 0.001, respectively, n = 117 (1), 57 (2), 57 (3), Figure 5E]. Thus, a structural gradient is present along the MN6/7b-Ib motor neuron with larger distal than proximal type Ib boutons. In addition, Brp is unevenly distributed in type Ib boutons of wt and rab3^rup NMJs.

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 [Ca²⁺]_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 brp⁶⁹ and syt^KD NMJs amplitude and kinetics of postsynaptic currents were comparable in distal and proximal boutons (n = 10 and 11 for brp⁶⁹ 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.

To test for a genetic interaction between Brp and Syt we analyzed non-allelic non-complementation (Yook et al., 2001). This genetic strategy tests for the ability of two recessive mutations to complement one another for a specific phenotype. We studied heterozygous animals carrying either one copy of the syt null allele Syt^AD4 (DiAntonio et al., 1993) or the brp null allele brp⁶⁹ (Kittel et al., 2006) and trans-heterozygous animals carrying both. We analyzed the structural gradient along the MN6/7b-Ib motor axon regarding AZ number and bouton dimensions (Figure 7). Whereas in heterozygous animals distal boutons contained more Brp puncta than proximal boutons (p < 0.001, n = 95 (1), 72 (2), 72 (3) for syt^AD4; p < 0.001, n = 51 (1), 40 (2), 40 (3) for brp⁶⁹), this gradient was lost in trans-heterozygous animals (9.8 ± 0.5, 10.0 ± 0.4, 11.0 ± 0.5, p > 0.05, n = 81 (1), 81 (2), 81 (3), Figure 7A). Interestingly, distal boutons of trans-heterozygous animals were still largest along the bouton chain (7.3 ± 0.4 μm², 5.5 ± 0.3 μm², 5.8 ± 0.3 μm², p = 0.003 and 0.014 for (2) and (3), Figure 7B). We conclude that regarding AZ distribution both mutations fail to complement one another, suggesting a direct interaction of Brp and Syt or an action of the two proteins in the same functional pathway.

To further investigate the impact of Brp and Syt on maintaining the structural gradient we again performed immunostainings (Figure 8). At brp⁶⁹ 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.

In a final set of experiments we employed dSTORM to image glutamatergic boutons. This localization microscopy technique substantially increases spatial resolution compared to conventional fluorescence light microscopy (Heilemann et al., 2008; van de Linde et al., 2011) and can provide quantitative insight into the nanoscopic organization of presynaptic AZs (Sauer, 2013; Ehmann et al., 2014, 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 μm² and 0.069 ± 0.002 μm², 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.