Calcium green

Fly stocks and genetics. Drosophila stocks were maintained at 22–25°C on normal food. Unless otherwise noted, all fly lines were obtained from the Bloomington Drosophila Stock Center or the Exelixis Collection (Harvard Medical School). UAS–RIM RNAi animals were obtained from the Vienna Drosophila Stock Center (stock GD15273). Standard second and third chromosome balancers and genetic strategies were used for all crosses and for maintaining mutant lines. The rim¹⁰³ allele was generated by imprecise excision of parental transposon P{EPgy2}Rim[EY05246] (insertion position: 13,710,797), 393 bp upstream of exon 16 (see Fig. 1A). For pan-neuronal expression, we used driver elav ^c155–Gal4 on the X chromosome (male larvae) in combination with UAS–dicer2 on the second chromosome (Dietzl et al., 2007 (link)). Unless noted, male and female larvae were used. Unless otherwise noted, the w¹¹¹⁸ strain was used as a wild-type (WT) control.
Electrophysiology. Sharp-electrode recordings were made from muscle 6 in abdominal segments 2 and 3 of third-instar larvae using an Axopatch 200B or a Multiclamp 700B amplifier (Molecular Devices) as described previously (Davis and Goodman, 1998 (link)). Two-electrode voltage-clamp recordings were performed with an Axoclamp 2B amplifier. The extracellular HL3 saline contained the following (in mM): 70 NaCl, 5 KCl, 10 MgCl₂, 10 NaHCO₃, 115 sucrose, 4.2 trehalose, 5 HEPES, and 0.4 (unless specified) CaCl₂. For acute pharmacological homeostatic challenge, larvae were incubated in Philanthotoxin-433 (PhTX; 10 or 20 μm; Sigma-Aldrich) for 10 min (Frank et al., 2006 (link)). EGTA-AM (25 μM in HL3; Invitrogen) was applied to the dissected preparation for 10 min. After EGTA application, the preparation was washed with HL3 for 5 min. The average single action potential (AP)-evoked EPSP amplitude (stimulus duration, 3 ms) or EPSC amplitude of each recording is based on the mean peak EPSP amplitude or EPSC amplitudes in response to 30 presynaptic stimuli unless specified. For each recording, we analyzed at least 100 miniature EPSPs (mEPSPs) to obtain a mean mEPSP amplitude value. Quantal content was estimated for each recording by calculating the ratio of EPSP amplitude/average mEPSP amplitude and then averaging recordings across all NMJs for a given genotype. EPSC data were analyzed in the same way.
The apparent size of the RRP was probed by the method of cumulative EPSC amplitudes (Schneggenburger et al., 1999 (link)), which was recently applied to the Drosophila NMJ (Hallermann et al., 2010 (link); Miśkiewicz et al., 2011 (link); Weyhersmüller et al., 2011 (link)). Muscles were clamped to −65 mV, and EPSC amplitudes during a stimulus train (60 Hz, 30 stimuli) were calculated as the difference between peak and baseline before stimulus onset of a given EPSC. The number of release-ready vesicles was obtained by back-extrapolating a line fit to the linear phase of the cumulative EPSC plot (the last 200 ms of a train) to time 0 (see Fig. 7A,B, bottom). The number of release-ready vesicles is then obtained by dividing the cumulative EPSC amplitude at time 0 by the mean mEPSC amplitude recorded in the same cell (see Fig. 7C, right). Because of initial facilitation/delayed depression of EPSC amplitudes during trains under conditions of reduced release probability (0.4 mM [Ca²⁺]_e), the RRP at low [Ca²]_e was assessed with longer trains (100 stimuli), and the RRP size estimate was based on a later linear phase of the cumulative EPSC data >1.2 s; see Fig. 7D). It is worth noting that the resulting RRP estimate at 0.4 mM [Ca²]_e may overestimate the total RRP as a result of “recovery from depression” (Schneggenburger et al., 1999 (link); Weyhersmüller et al., 2011 (link)).
For fluctuation analysis (see Fig. 8), the mean EPSC amplitude (I) and the EPSC amplitude variance of each synapse at each extracellular calcium concentration ([Ca²]_e; 0.3, 1, and 3 mM; [Mg²]_e, 10 mm) was based on 40–150 consecutive EPSCs (interstimulus interval, 5 s). EPSC amplitude variance was calculated according to previous reports (Meyer et al., 2001 (link); Scheuss and Neher, 2001 ; Scheuss et al., 2002 (link)), and the quantal parameters N and q were obtained by fitting the EPSC variancemean data of each synapse with a parabola [Var(I)=I²/N + qI] that was constrained to pass through the origin. N and q values were then averaged across cells. The mean coefficients of variation of mEPSC amplitudes of all groups were similar (data not shown), and values for q and N were not corrected for variability in mEPSC amplitude distributions (Brown et al., 1976 (link); Silver et al., 1998 (link); Scheuss and Neher, 2001 ) or latency fluctuations (“jitter”) of individual quantal events (Taschenberger et al., 2005 (link); Weyhersmüller et al., 2011 (link)).
Ca²⁺ imaging. Ca²⁺ imaging experiments were done as described by Müller and Davis (2012) (link). Third-instar larvae were dissected and incubated in ice-cold, Ca²⁺-free HL3 containing 5 mm Oregon-Green 488 BAPTA-1 (OGB-1) (hexapotassium salt; Invitrogen) and 1 mm Alexa Fluor 568 (Invitrogen). After incubation for 10 min, the preparation was washed with ice-cold HL3 for 10–15 min. Single action-potential evoked spatially averaged Ca²⁺ transients were measured in type-1b boutons synapsing onto muscle 6/7 of abdominal segments A2/A3 at an [Ca²⁺]_e of 1 mm using a confocal laser-scanning system (Ultima; Prairie Technologies) at room temperature. Excitation light (488 nm) from an aircooled krypton–argon laser was focused onto the specimen using a 60× objective (1.0 NA; Olympus), and emitted light was detected with a gallium arsenide phosphide-based photocathode photomultiplier tube (Hamamatsu). Line scans across single boutons were made at a frequency of 313 Hz. Fluorescence changes were quantified as ΔF/F = (F(t) — F_baseline)/(F_baseline — F_background), where F(t) is the fluorescence in a region of interest (ROI) containing a bouton at any given time, F_baseline is the mean fluorescence from a 300 ms period preceding the stimulus, and F_background is the background fluorescence from an adjacent ROI without any indicator-containing cellular structures. One synapse (4–12 boutons) was imaged per preparation. The average Ca²⁺ transient of a single bouton is based on 8–12 line scans. Experiments in which the resting fluorescence decreased by >15% and/or which had an F_baseline > 650 a.u. were excluded from analysis. Data of experimental and control groups were collected side by side. The Ca²⁺ indicator was not saturated by single AP stimulation because repetitive stimulation induced an additional increase in peak ΔF/F (20 ms interstimulus interval; data not shown). The intraterminal Ca²⁺ indicator concentration (~50 μm was roughly approximated by an in vitro calibration (Müller and Davis, 2012 (link)).
Data analyses. Electrophysiology data and Ca²⁺ imaging data were analyzed with custom-written routines in Igor Pro 6.22 (Wavemetrics), and spontaneous mEPSPs were analyzed with Mini Analysis 6.0.0.7 (Synaptosoft). Ca²⁺ imaging data was acquired with Prairie View. Deconvolution microscopy data (see Fig. 2A,B) were acquired and analyzed with Intelligent Imaging Innovations (3i) software. Structured-illumination (SIM) data (Fig. 2C–E) was acquired with ZEN software (Carl Zeiss) and analyzed with custom-written macros in NIH ImageJ/Fiji (W. S. Rasband, National Institutes of Health, Bethesda, MD; Schindelin et al., 2012 (link)) and Igor Pro. All results are reported as average ± SEM. Statistical significance was assessed by Student’s t test unless otherwise specified.
Quantitative RT-PCR. Quantitative RT-PCR was performed as described by Berquist et al. (2010) (link). Primer probes were designed and developed by Applied Biosystems. The CNS was removed from 25 third-instar larvae per sample (three samples per genotype). Total RNA was isolated from each sample using the standard Trizol protocol. A DNase digestion removed potential DNA contamination (RQ1 RNase-free DNase; Promega). RT was performed (Taqman reverse transcription reagents; Applied Bioscience) using random hexamers and 1 μg of total RNA. A no-RT control was performed for each sample. Purified cDNA was used as a template in 30 μl of PCR reaction (TaqMan Universal PCR Master Mix, no AmpErase UNG; Applied Biosystems). This 30 μl reaction was divided into three 10 μl triplicates. In addition, one 10 μl no-RT reaction was used for each sample. The ABI Prism 7900 was used for all PCRs. Cycle threshold (C_T) was determined by automated threshold analysis using SDS2.3 software according to the instructions of the manufacturer (Applied Biosystems). Comparative levels (between WT and mutant animals) were determined using the ΔΔC_T method (Applied Biosystems User Bulletin 2). To determine whether the two amplification reactions have the same PCR efficiency, ΔC_T (C_T of experimental gene — C_T of reference gene) values are determined across the serial dilutions and plotted against the log of the cDNA dilution. Briefly, the ΔΔC_T method is as follows. ΔC_T values are determined as explained above. Next, experimental animal (mutants) ΔC_T values were subtracted from control animal (WT) ΔC_T values to give the ΔΔC_T. Finally, using the equation 2^([—]ΔΔC_T) × 100, the percentage expression of each gene in experimental compared with control animals was calculated. Each experimental animal sample was compared to each WT sample (Applied Biosystems User Bulletin No. 2).
Synapse morphology. Third-instar larval preparations were fixed for 2 min in Bouin’s fixative (100%; Sigma-Aldrich) or for 15 min in PFA (4% in PBS) and incubated overnight at 4°C with primary antibodies. The following primary antibodies were used at the indicated dilutions: mouse anti-Bruchpilot (Brp), 1:100 (nc82; Kittel et al., 2006 (link)); and rabbit anti-Dlg, 1:5000. Alexa Fluor-conjugated secondary antibodies and Cy3-conjugated anti-HRP were used at 1:200 and 1:800, respectively (Jackson ImmunoResearch; Invitrogen), and applied for 2 h at room temperature. Larval preparations were mounted in Vectashield (Vector Laboratories). An Axiovert 200 inverted microscope (Carl Zeiss), a 100× (1.4 NA) Plan Apochromat objective (Carl Zeiss) and a cooled CCD camera (CoolSNAP HQ; Roper Scientific) were used for deconvolution microscopy, and data were analyzed as described previously (Pielage et al., 2008 (link)) (see Fig. 2A,B).
For SIM imaging, we used an ELYRA PS.1 system (Carl Zeiss) with an inverted LSM-710 microscope, a 63× (1.4 NA) Plan-Apochromat objective (Carl Zeiss), and an Andor iXon 885 EMCCD camera. Lateral resolution was ~110 nm, and axial resolution was ~300 nm. Z-stacks of whole NMJs at muscle 4 were taken with oversampling in xy (40 × 40 nm pixel size) and z (110 nm step size). Individual Brp puncta were identified with a threshold-based mask applied to the maximum projection of a Z-stack (Fouquet et al., 2009 (link)), and confluent puncta were removed manually. A fluorescence intensity line profile (1.2 μm long, 1 pixel wide) was obtained along the major and minor axis of a bounding ellipse that was fitted to each punctum. Diameter analysis was restricted to Brp puncta with a planar orientation with respect to the focal plane. These puncta were detected by a local minimum around the centroid of the ellipse in both line profiles. The maximum “peak-to-peak diameter” of a Brp punctum was calculated as the distance between the peaks of the line profile along the major axis of the ellipse. The “diameter at halfmaximum” was computed as the maximum distance between two points at 50% of the peak of the same profile (see Fig. 2E).