Abstract
At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. Here, we identified a hypomorphic allele, brpnude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. Our results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression.
Introduction
The rate at which sensory information can be transmitted across synapses is an important factor that limits rapid and precise information processing in the nervous system. The mechanisms that operate in the presynaptic terminals to release neurotransmitter at AZs at high repetition rates are currently intensely investigated (Atwood and Karunanithi, 2002; Zucker and Regehr, 2002; Kidokoro et al., 2004; Neher and Sakaba, 2008). AZs are decorated with electron-dense material of varying shape and size depending on synapse type and species (Zhai and Bellen, 2004).
Recently, the CAST/ERC-family member Bruchpilot (BRP), a coiled-coil rich protein of nearly 200 kDa, was identified via its localization to Drosophila AZs. BRP was shown to be crucial for cytomatrix formation, proper Ca2+ channel clustering within AZs, and efficient neurotransmitter release at the Drosophila neuromuscular junction (Kittel et al., 2006; Wagh et al., 2006). Furthermore, BRP is a direct component of the electron-dense cytomatrix (T-bar) and adopts an elongated conformation, with its N terminus (N-term) facing Ca2+ channels at the membrane and its C-term reaching into the cytoplasm (Fouquet et al., 2009).
Materials and Methods
Genetics.
Larvae were raised at 25°C [cacophony (Cac) RNA interference (RNAi) larvae and their controls were raised at 29°C] in bottles or on apple agar plates (for high-pressure freezing), and the following genotypes were used: Bruchpilot studies: brpnude or brp1.3 or brp5.45/df(2R)BSC29, ok6-GAL4 (Fouquet et al., 2009), controls: +/ok6-GAL4. Cacophony images: ok6-GAL4, UAS-CacGFP/+ and brpnude/df(2R)BSC29, ok6-GAL4; UAS-CacGFP/+. Cacophony RNAi studies: UAS-Cac RNAi (Transformant ID 5551) (Dietzl et al., 2007)/+;elav-GAL4/+, control: elav-GAL4/+.
Stimulated emission depletion microscopy.
Stimulated emission depletion (STED) microscopy experiments were performed essentially as described by Kittel et al. (2006), using the commercially available Leica TCS STED microscope (Leica Microsystems). Secondary antibodies were conjugated to the Atto 647N dye (AttoTech). Atto 647N fluorophores were excited at a wavelength of 635 nm and depleted at 750 nm. Images were acquired using APD detectors within a range of 645–715 nm. Image processing was performed using the Imspector software (Max-Planck Innovation) by applying a linear deconvolution at single STED slices.
To assess Ca2+ channel clustering, neuromuscular junctions were stained with M-α-GFP (Invitrogen) and G-α-M-Atto647N to visualize Ca2+ channels (CacGFP). Additionally, serving as a reference, postsynaptic glutamate receptors were labeled with primary Rb-α-DGluRIID and secondary Sheep-α-Rb-Cy3 (Invitrogen) antibodies and imaged via conventional confocal microscopy. From such images, AZs were selected that appeared planar to the optical slice (i.e., the AZ membrane is parallel to the focal slice). With Mathematica 5.0 (Wolfram Research), each channel (CacGFP and GluRIID) of each image of a planar AZ was automatically fitted with a two-dimensional Gaussian function. The peak of these Gaussians was used to align the CacGFP signal either with itself or with the GluRIID signal for subsequent averaging. The average images were again fitted with two-dimensional Gaussian functions and both horizontal and vertical intensity profiles through the peak of the two-dimensional Gaussian functions were averaged and fitted with one-dimensional Gaussian functions (see Fig. 2B).
Electron microscopy.
Electron microscopy and conventional embedding was performed as previously described (Fouquet et al., 2009) on late third-instar Drosophila larvae. The number of docked vesicles was determined along the whole synapse showing the typical close apposition of electron-dense presynaptic and postsynaptic membrane. We counted only vesicles without any discernible distance between vesicle and AZ membrane.
Electrophysiology.
Two-electrode voltage-clamp (TEVC) recordings of EPSCs were obtained at room temperature from late third-instar male Drosophila larvae (ventral longitudinal muscle 6, segments A2 and A3), essentially as previously described (Kittel et al., 2006). For the TEVC experiments with brpnude, brp1.3, and brp5.45 mutants, both mutants and control animals carried a copy of ok6-GAL4 to ensure comparability with a previous study (Kittel et al., 2006). The composition of the extracellular hemolymph-like saline (HL-3) was as follows (in mm): NaCl 70, KCl 5, MgCl2 20, NaHCO3 10, trehalose 5, sucrose 115, HEPES 5, CaCl2 1 or as indicated, and pH adjusted to 7.4. Recordings were made from cells with an initial resting membrane potential between −50 and −70 mV (holding potential at −60 mV) using intracellular electrodes with resistances of 10–32 MΩ, filled with 3 m KCl. Train stimulation protocols consisted of 100 pulses applied at 60 Hz. The recovery was assessed by evoking APs at (in ms following the last pulse in the train): 25, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, and 100,000 (Wu et al., 2005). EPSCs reflect the compound response to stimulation of both motoneurons innervating muscle 6, and care was therefore taken to ensure their stable recruitment. Infrequently observed recruitment failures were linearly interpolated. Only cells that recovered at least 70% of their initial EPSC amplitude following tetanic stimulation were included in the analysis. The recordings were analyzed with pClamp 9 (Molecular Devices), and the peak amplitude was determined as the difference between the peak value of the EPSC and the baseline value before onset of that EPSC. The steady-state EPSC amplitudes (see Fig. 3D) were evaluated for each experiment as the average of the last 25 EPSC amplitudes in the train and normalized to the average steady-state EPSC amplitude of the corresponding control for either brp or Cac RNAi experiments. The time constants of the fast and slow components of recovery (see Fig. 3E,F) were determined from monoexponential functions fitted to the average fast and slow component of recovery and weighted with the errors of the average data with Igor Pro 6.1 (Wavemetrics). The estimated errors of the fit parameters were taken from Igor Pro (which are based on the square roots of the diagonal elements of the covariance matrix). From the time constants, the SEs, and the number of experiments, statistical comparisons were performed with Student's t test, and the error bars of the normalized values were calculated according to Gaussian error propagation.
Statistical analysis.
The nonparametric Mann–Whitney rank sum test was used for statistical analysis if not stated otherwise. The data are reported as mean ± SE, n indicates the sample number, and p denotes the significance (*p < 0.05, **p < 0.01, and ***p < 0.001).
Results
Impaired vesicle tethering at AZs of a Bruchpilot mutant (brpnude) lacking the last 17 C-terminal amino acids
In a chemical [ethyl methyl sulfonate (EMS)] mutagenesis screen, a novel brp allele was identified with a premature STOP codon at amino acid position 1724 (brp5.38, hereafter brpnude) of 1740 aa. While in the previously published mutants brp5.45 and brp1.3 a significant proportion of the C-terminal amino acids are absent (50% and 30%, respectively; STOP codons at position 867 or 1390) (Fouquet et al., 2009), brpnude lacks only the last 1% (Fig. 1A,C). Nevertheless, all three mutants showed significantly reduced survival rates and motor abilities as adult flies (Fig. 1B).
Impaired vesicle tethering at AZs of a bruchpilot mutant (brpnude) lacking the last 17 C-terminal amino acids. A, Chemical induction of premature stop codons resulted in truncated versions of the BRP protein (BRP; dark blue; boxes indicate coiled-coil domains) of ∼99% (brpnude; gray), of ∼70% (brp1.3; green), and of ∼50% length (brp5.45; blue). Below, Illustration of the BRP protein (blue) within the AZ (red: Ca2+ channels; gray: synaptic vesicle) and the corresponding positions of the truncation (arrows) (Fouquet et al., 2009). B, All three BRP mutants (brpnude, brp1.3; brp5.45) showed severely impaired survival rates and walking skills. C, In the brpnude allele, a single base mutation at the C-terminal position 1724 leads to a premature stop codon and a BRP protein (BRPnude) lacking the last 17 of 1740 aa. D, STED images of neuromuscular junctions stained with a C-terminal BRP antibody (green; BRPNc82) and simultaneous confocal images of an N-terminal BRP antibody (magenta; BRPN-term). The distribution of both antibody signals appears unaltered at brpnude synapses (right) compared to controls (left). Arrows and arrowheads indicate planar and vertical AZs, respectively. E, Examples of conventionally embedded AZs of control and brpnude. Note fewer tethered vesicles in brpnude. F, Height and length of the platform of the electron-dense cytomatrix (T-bar) for control and brpnude. G, The number of docked vesicles per AZ section for control and brpnude (n = 22 and 25 AZs, respectively) and the number of vesicles found within three shells (see inset) each of 50 nm thickness surrounding the AZ (n = 20 and 31 AZs, respectively). Scale bars: D, 1 μm; E, 200 nm.
At neuromuscular junctions of brpnude, the reactivities of antibodies directed against the C-term (BRPNc82; Wagh et al., 2006) and the N-term (BRPN-term) (Fouquet et al., 2009) of BRP are still present (Fig. 1D), and the number of AZs per junction is normal (quantified as BRPNc82 spots per junction; 553 ± 27 and 518 ± 28 for control and brpnude; njunction = 6 and 6; p = 0.24). Furthermore, high-resolution light microscopic images (STED microscopy) (Hell, 2007) of BRPNc82 acquired simultaneously with confocal images of BRPN-term appeared unchanged compared to controls. Especially, the previously described ring-like distribution of Nc82 reactivity (Kittel et al., 2006) is preserved at brpnude synapses (Fig. 1D). The normal distribution of C- and N-terminal signals at brpnude AZs indicates that the missing 1% of BRP does not alter the overall structure of the cytomatrix. In fact, an electron microscopic analysis of the ultrastructure of brpnude synapses revealed normal amounts of AZ cytomatrix and an ordinary height and platform length of dense bodies (T-bars) (Fig. 1E,F). Furthermore, the diameter of the synaptic vesicles as well as the total number of synaptic vesicles per bouton section were unchanged in brpnude (diameter: 36 ± 1.7 and 35 ± 0.3 nm for control and brpnude, nvesicles = 220 and 243 (10 larvae each), respectively; p = 0.9; number: 114 ± 24 and 131 ± 7 SV/μm2 for control and brpnude, nbouton-section = 6 and 6, respectively; p = 0.2). Finally, the number of docked vesicles per AZ section was not significantly different between brpnude and controls (1.8 ± 0.2 and 1.4 ± 0.3 for control and brpnude, nAZ = 21 and 24, respectively; p = 0.4) (see Fig. 1G; for definition of docked vesicles, see Materials and Methods). However, T-bars at brpnude AZs were bare of vesicles (Fig. 1E). A quantification of the number of vesicles within three shells of each 50 nm thickness surrounding the AZ revealed a significant reduction in the average number of vesicles near the T-bar at brpnude synapses (Fig. 1G). These data indicate that while the basic ultrastructure of the cytomatrix is unaltered at brpnude AZs, vesicle tethering is specifically impaired.
Normal Ca2+ channel clustering and basal release at brpnude synapses
Using STED microscopy, we tested whether Ca2+ channel clustering is affected at brpnude synapses. STED images of GFP-labeled Ca2+ channels (CacGFP) were acquired simultaneously with confocal images of postsynaptic glutamate receptors (GluRIID) to identify the position of AZs (opposite GluRIID patches) independently of Ca2+ channel clustering (Fig. 2A). Fitting Gaussian functions to the intensity profiles of average images revealed a normal Ca2+ channel distribution at brpnude synapses (Fig. 2B). Comparable results were obtained when the Cac signals were aligned with themselves. With this method, the delocalization of Ca2+ channels in brp null mutants (Kittel et al., 2006) could clearly be resolved (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). Thus, we isolated a brp allele lacking only the last 1% of C-terminal amino acids, which severely affects vitality but not the clustering of AZ Ca2+ channels.
Normal Ca2+ channel clustering and basal release at brpnude synapses. A, GluRIID labels (magenta, confocal) were used to quantify the Ca2+ channel (CacGFP, green, STED) clusters in control animals (top row) and brpnude mutants (below). The Cac signals at synapses that appeared ideally planar (n = 16 each) were averaged after alignment with the postsynaptic GluRIID signal (right panels) or themselves (center panels). While the example images were scaled up individually, all four averages were scaled up with the same factor. B, Intensity profiles of the average Cac images in black for controls and in gray for brpnude mutants with SE bars and the corresponding Gaussian fits (standard deviations given by σ) in gray. C, Examples of EPSCs elicited at 0.2 Hz in control (black), brpnude (gray), brp1.3 (green), and brp5.45 mutants (blue; average of 10 each) in 1.0 mm Ca2+. The average peak EPSC amplitudes and rise and decay kinetics were normal in brpnude mutants. In brp1.3 and brp5.45 mutants EPSC amplitudes were reduced and rise times increased (n = 22, 18, 4, and 12 for control, brpnude, brp1.3, and brp5.45, respectively). D, Example traces of mEPSCs of control (black), brpnude (gray), brp1.3 (green), and brp5.45 mutants (blue). The average mEPSC amplitudes were normal in brpnude, brp1.3, and brp5.45 mutants (n = 6, 6, 4, and 6 for control, brpnude, brp1.3, and brp5.45, respectively). Scale bar in A, 250 nm.
Next, we tested the functional consequences of defective vesicle tethering. EPSCs evoked at 0.2 Hz at brpnude synapses did not differ in terms of their peak amplitude, rise time, or decay time constant from those of controls (Fig. 2C). In brp1.3 and brp5.45 mutants, with poorly clustered Ca2+ channels (Fouquet et al., 2009), the EPSC amplitudes were reduced to 30 ± 8% (n = 4) and 10 ± 8% (n = 12) of control amplitudes, respectively (Fig. 2C). The amplitude of miniature EPSCs was not significantly affected in any of these three mutants (Fig. 2D). These data indicate that basal release (at 0.2 Hz) requires Ca2+ channel clustering, but not vesicles tethered to the cytomatrix.
Tethering vesicles to the active zone dense body prevents synaptic depression
To investigate the impact of vesicle tethering on short-term plasticity, we analyzed synaptic transmission during paired-pulse stimulation (Fig. 3A). Strikingly, the amplitude of the second EPSC was significantly reduced at short interpulse intervals of 10 and 30 ms (Fig. 3B). This depression decayed with a time constant of 19 ms. Next, synaptic transmission was analyzed during a train of 100 stimuli at 60 Hz (Fig. 3C) (Hallermann et al., 2010). The depression during the train was stronger in brpnude as quantified by a reduction in the steady-state EPSC amplitude at the end of the train to 35 ± 3% compared to 52 ± 5% in controls (n = 21 and 20 for control and brpnude mutants, respectively) (Fig. 3C,D). Consistent with the altered paired-pulse ratio, the amplitude of the second EPSC in the train was already significantly reduced to 80 ± 10% at brpnude synapses (n = 21 and 20).
Tethering vesicles to the AZ dense body prevents synaptic depression. A, Examples of EPSCs elicited by a paired-pulse protocol with 30 ms interpulse interval in control (black) and brpnude (gray). B, Average paired-pulse ratios plotted versus interpulse interval in 1.0 mm Ca2+ for control (black, n = 12) and brpnude (gray, n = 9; asterisk indicates significant differences) superimposed with exponential fits (time constants are indicated). Inset, 0.6 mm Ca2+. C, Average peak EPSC amplitudes during the 60 Hz train for control (black, n = 21) and brpnude (gray, n = 20). Inset, Average amplitudes of the first 5 EPSCs of the train. The steady-state amplitude (average of EPSC amplitudes marked by the bracket) was significantly reduced in brpnude mutants compared to controls. D, Steady-state amplitudes normalized to the steady-state amplitude of the respective control, for brpnude (gray), brp1.3 (green), brp5.45 (blue), and Cac RNAi synapses (red) in extracellular Ca2+ concentrations that lead to normal (“rescued”) basal EPSC amplitudes ([Ca2+]rescue) (see supplemental Fig. S2, available at www.jneurosci.org as supplemental material). E, Average peak EPSC amplitudes during the first rapid component of recovery after the train (τ1) for control (black, n = 21) and brpnude (gray, n = 20) fitted exponentially (lines). Time constants with SEs are indicated. Bottom, The time constants of the first component of recovery normalized to the corresponding time constant of the control (color code as in D). F, Corresponding average peak EPSC amplitudes during the second slower component of recovery after the train (τ2) with corresponding average data.
If sustained release is impaired in brpnude mutants because fewer vesicles are tethered to the cytomatrix, then sustained release in brp1.3 and brp5.45 mutants, both of which show severely impaired cytomatrices (Fouquet et al., 2009), should be impaired, too. Indeed, investigations of sustained release in brp1.3 and brp5.45 mutants revealed stronger synaptic depression than in controls (supplemental Fig. S2A,C, available at www.jneurosci.org as supplemental material). To analyze whether the stronger depression in brp1.3 and brp5.45 mutants is simply the consequence of impaired Ca2+ channel clustering, we sought to compensate (“rescue”) the reduced Ca2+ channel density by increasing the extracellular Ca2+ concentration. In high Ca2+ (2 mm and 2.5 mm for brp1.3 and brp5.45 mutants, respectively), their EPSC amplitudes were comparable to those of controls in 1 mm Ca2+. However, depression during sustained release was still pronounced (Fig. 3D; supplemental Fig. S2B,D, available at www.jneurosci.org as supplemental material).
Since vesicle recruitment critically depends on the intracellular spatiotemporal Ca2+ dynamics (Neher and Sakaba, 2008), we performed control experiments by reducing the density of Ca2+ channels genetically and tested whether subsequent elevations of extracellular Ca2+ could rescue sustained release. To this end, transgene-mediated RNAi directed against the α1 subunit of the Drosophila Ca2+ channel (Cac) was performed, resulting in a reduction of EPSC amplitudes by ∼50%. Extracellular Ca2+ was then elevated to 1.5 mm to obtain control EPSC amplitudes. However, in contrast to brp1.3 and brp5.45 mutants, sustained release was unaltered by Cac RNAi (Fig. 3D; supplemental Fig. S2E, available at www.jneurosci.org as supplemental material).
Next, we addressed whether the enhanced depression during sustained high-frequency transmission at brpnude synapses is accompanied by alterations in the kinetics of recovery from depression. Therefore, the recovery from synaptic depression after a train was investigated with test stimuli of increasing intervals following the train (Fig. 3E,F) (Wu et al., 2005; Hallermann et al., 2010). A biphasic recovery with time constants of τ1 = 50 ms and τ2 = 6.1 s was found in controls. In brpnude mutants, the first component was slower (114 ms), while the second component was unaltered (5.6 s) (Fig. 3E,F). Consistently, the first component of recovery was also slower in brp1.3 and brp5.45 mutants with Ca2+ concentrations that rescued the basal EPSC amplitude. In contrast to the normal second component of recovery at brpnude synapses, the second component of recovery in brp1.3 and brp5.45 mutants was also significantly slower (Fig. 3E,F; supplemental Fig. S2, available at www.jneurosci.org as supplemental material). Finally, in experiments with Cac RNAi, which serve as a control for the approach of elevating Ca2+, both components were normal (Fig. 3E,F; supplemental Fig. S2, available at www.jneurosci.org as supplemental material). In summary, these data indicate that the basal EPSC amplitude (cf. Fig. 2) as well as the second component of recovery rely on adequate Ca2+ channel clustering (impaired in brp1.3 and brp5.45) and that high-frequency sustained release as well as the first component of recovery also rely on proper vesicle tethering at the AZ (selectively impaired in brpnude).
Discussion
To our knowledge, the specific impairment of vesicle tethering reported here delivers the first direct demonstration that efficient sustained release relies on the ability of the AZ to tether vesicles. While the overall AZ structure, including the distribution of Ca2+ channels, was unaffected, the impairment of vesicle tethering provoked pronounced synaptic depression and a slowed first component of recovery.
The C-terminal half of BRP consists of ∼1000 aa of essentially contiguous coiled-coil sequence (Wagh et al., 2006), reminiscent of Golgi/ER-resident tethering factors such as, e.g., GM130 (Lupashin and Sztul, 2005). These coiled-coils typically form rod-like structures, where 100 aa residues extend ∼15 nm when dimerized, and proteins such as Uso1p (Yamakawa et al., 1996) extend over 150 nm (Lupashin and Sztul, 2005). These rod-like proteins are believed to act before SNARE protein assembly by forming contacts between membranes at a distance, thereby increasing the specificity or efficiency of the initial attachment of vesicles (tethering) (Guo et al., 2000). We have provided morphological and functional evidence that BRP filaments tether vesicles, and thus further mechanistic comparisons between AZ and Golgi/ER trafficking, e.g., concerning the role of small GTPases, might well be informative.
The C-terminal half of BRP is very highly conserved in insects but not elsewhere (Wagh et al., 2006). Interestingly, the Drosophila genome does not appear to encode homologs of the vertebrate AZ components Piccolo and Bassoon (Wagh et al., 2006), which are key regulators of the vertebrate cytomatrix (Khimich et al., 2005). At central vertebrate synapses, CAST and Bassoon immunoreactivities (closer and further from the AZ membrane, respectively) were recently found to be associated with filaments that may connect vesicles to the AZ (Siksou et al., 2007). It is tempting to speculate that at AZs of central vertebrate synapses, CAST associates with coiled-coil domain proteins, such as bassoon, to perform the dual functions of Ca2+ channel clustering and vesicle tethering executed by the N-terminal and the C-terminal domains of BRP, respectively.
How synapses manage to repetitively release transmitter with high precision is intensely investigated. Vesicles tethered to electron-dense bodies may represent a reservoir of vesicles required for sustained release (Zhai and Bellen, 2004). Consistent with this hypothesis, synaptic stimulation provokes depletion of vesicles tethered at dense bodies (LoGiudice et al., 2008; Jackman et al., 2009). While the supply of vesicles appears rate limiting during the train and the first component of recovery (Saviane and Silver, 2006), the maturation of vesicles closer to Ca2+ channels appears rate limiting during the second component of recovery [cf. brp1.3 and brp5.45 (Kittel et al., 2006; Fouquet et al., 2009)]. One may argue that the rapid component of depression observed at brpnude synapses (Fig. 3A,B) could be partially attributed to fewer docked vesicles (though not significantly; cf. Fig. 1G) with a higher initial release probability. However, a functional estimation of the number of readily releasable vesicles using back-extrapolation from the cumulative EPSC amplitudes in the trains (Schneggenburger et al., 1999) revealed similar numbers of readily releasable vesicles in brpnude and controls (supplemental Fig. S3, available at www.jneurosci.org as supplemental material). Finally, we would like to point out that the C-term of BRP could be involved in endocytotic mechanisms, which have been shown to be crucial for sustained release (Koenig et al., 1998; Kawasaki et al., 2000; Dickman et al., 2005; Hosoi et al., 2009; Yao et al., 2009). Novel techniques have begun to address the spatial organization of local vesicle reuse within active zones (Zhang et al., 2009). It will have to be clarified via which routes vesicles move within active zones and in which direction Bruchpilot steers their translocation.
Footnotes
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to S.J.S. (Exc 257, SI849/2-1, TP A16/SFB 406, TP B23/SFB 581) and to M.H. (HE 2621/4-2 and TP B27/SFB 581), and by formel.1 grants to S.H. and R.J.K. from the Medical Faculty of the University of Leipzig. R.J.K. is supported by the DFG Emmy Noether-Program (KI 1460/1-1). We acknowledge the VDRC for the cacophony RNAi line.
- Correspondence should be addressed to either of the following: Manfred Heckmann, Physiologisches Institut, Röntgenring 9, 97070 Würzburg, Germany, heckmann{at}uni-wuerzburg.de; or Stephan J. Sigrist, Institute for Biology/Genetics, Free University Berlin, Takustrasse 6, 14195 Berlin, Germany, E-mail: stephan.sigrist{at}fu-berlin.de
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