Figure 2. Characterization of short-term plasticity at the Drosophila melanogaster NMJ.

Two-electrode voltage clamp recordings of AP-evoked synaptic transmission in muscle 6 NMJs (genotype: Ok6-GAL4/+ (Ok6-Gal4/II crossed to w[1118])). Left panel (A, C, E) shows example traces from one cell. Right panel (B, D, F) shows quantification across cells. (A) Representative eEJC traces from a single cell measured at different Ca²⁺ concentrations (0.75–10 mM). (B) Average eEJC₁ amplitudes and SD from six animals as a function of extracellular Ca²⁺ concentration. (C) Representative eEJC traces of paired pulse paradigm (10 ms inter-stimulus interval, normalized to eEJC₁) from single cell measured at different Ca²⁺ concentrations (0.75–10 mM). While STF can be seen at the two lowest extracellular Ca²⁺ concentrations (0.75 and 1.5 mM), the cell exhibits STD for extracellular Ca²⁺ concentrations of 3 mM or more. Insert (gray background) shows calculation of eEJC₂. An exponential function was fitted to the decay to estimate the baseline for the second response (see Figure 1—figure supplement 1 and Materials and methods for details). (D) Mean and SD of PPR values (6 cells from six animals) at different Ca²⁺ concentrations. (E) Experiment to assess variance of repeated synaptic responses in a single cell. eEJC₁ traces in response to nine consecutive AP stimulations (10 s interval) are shown (orange lines) together with the mean eEJC₁ response (black line) at different extracellular Ca²⁺ concentrations (0.75–10 mM, see Materials and methods). (F) Plot of mean eEJC₁ variance as a function of the mean eEJC₁ amplitude across 6 cells from six animals for each indicated Ca²⁺ concentration. The curve shows best fitted parabola with intercept forced at (0,0) (Var = −0.0061*<eEJC₁>²+0.6375 nA*<eEJC₁>, corresponding to n_sites = 164 and q = 0.64 nA when assuming a classical binomial model (Clements and Silver, 2000), see Materials and methods). For the variance-mean relationship of the single cell depicted in Figure 2E , please refer to Figure 2—figure supplement 2. Experiments were performed in Ok6-Gal4/+ 3^rd instar larvae, often used as a control genotype for experiments using cell-specific driver lines. Separate experiments were performed to ensure that this genotype showed similar synaptic responses and STP behavior as wildtype animals (Figure 2—figure supplement 3). Used genotype: Ok6-Gal4/II crossed to w[1118]. Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes. Data points depict means, error bars are SDs across cells except in (F), where error bars show SEM. Raw data corresponding to the depicted graphs can be found in the accompanying source data file (Figure 2—source data 1). Scripts for analysis of recorded traces are found in accompanying source data zip file (Figure 2—source data 2). Raw traces from paired-pulse experiments summarized in Figure 2 and Figure 2—figure supplements 2 and 3 can be found in Figure 2—source data 2; Figure 2—figure supplement 1—source data 1; Figure 2—figure supplement 3—source data 1. Estimation of eEJC₂ amplitudes and fitting of a smooth mEJC function (used in simulations, see Materials and methods) are illustrated in Figure 2—figure supplement 1.

Figure 2—figure supplement 1. Illustration of analysis of experimental electophysiological data.

(A) Analysis of eEJCs. The eEJC₁ amplitude is determined as the first minimum within 10 ms of the stimulus. The eEJC₂ amplitude is determined from the baseline found by exponential extrapolation of the eEJC₁ decay. (B) The mean mEJC from experimental recordings and the best fit used for convolution (see Materials and methods). Used genotype: Ok6-Gal4/II crossed to w[1118]. Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes.

Figure 2—figure supplement 2. Illustration of fluctuation analysis (quantification across cells shown in Figure 2F) in a single representative cell.

(A) Example traces showing nine individual stimulation sweeps (orange) per Ca²⁺ concentration (0.75–10 mM). Averages of single sweeps are shown in black. (B) Quantification of eEJC amplitudes of single sweeps (orange) at different Ca²⁺ concentrations. Scattering of these values illustrates the variance of eEJC amplitudes between individual sweeps. Average eEJC amplitudes per Ca²⁺ concentration are indicated in black, error bars show SDs. Average amplitudes (and their variance) shown here were used in C. (C) Variances of eEJC amplitudes in this cell (from nine repetitions per Ca²⁺ concentration, indicated in blue) plotted as a function of the mean eEJC amplitude. A parabola can be fitted to the data points (forced through (0,0) intercept, see Materials and methods for futher information and exact genoytpes). Used genotype: Ok6-Gal4/II crossed to w[1118]. Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes.

Figure 2—figure supplement 3. Electrophysiological comparison of synaptic transmission in wildtype (w[1118], +/+) (black) and Ok6-Gal4/+ (orange) flies.

Electrophysiological recordings of muscle 6 NMJs upon a paired-pulse stimulation (10 ms inter-stimulus interval) at 0.75 mM (A-D) and 1.5 mM Ca²⁺(E-H). (A) Representative example traces for eEJC₁ from single cells in +/+ or Ok6-Gal4/+. (B) Quantification of eEJC₁ amplitudes in +/+ (n = 6 cells from three animals) and Ok6-Gal4/+ (n = 8 cells from four animals), averages and SDs across cells are indicated. (C) Representative normalized eEJC example traces of paired pulse (10 ms inter-stimulus interval) responses in representative cells of +/+ and Ok6-Gal4/+ animals. (D) Quantification of paired pulse ratios (PPRs) in +/+ (n = 6 cells from three animals) and Ok6-Gal4/+ (n = 8 cells from four animals), averages across cells and SDs are indicated. (E) Representative example traces for eEJC₁ from single cells of +/+ or Ok6-Gal4/+ animals. (F) Quantification of eEJC₁ amplitudes in +/+ (n = 5 cells from three animals) and Ok6-Gal4/+ (n = 5 cells from three animals), averages and SDs across cells are indicated. (G) Normalized eEJC example traces of paired pulse (10 ms inter-stimulus interval) from single cells in +/+ and Ok6-Gal4/+ animals. (G) Quantification of PPRs in +/+ (n = 5 cells from three animals) and Ok6-Gal4/+ (n = 5 cells from three animals), averages across cells and STDs are indicated. Used genotypes: Ok6-GAL4/+ (Ok6-Gal4/II crossed to w[1118]) and +/+: w[1118]. Materials and methods section ‘Fly husbandry, genotypes and handling’ lists all exact genotypes.